GRE Subject Tests: Biochemistry, Cell

and Molecular Biology

2014\2015
Afnan haj yahia
E-mail:afnan.haj-92@hotmail.com
0526205771

F cont.

Alanine scanning

Fast parallel proteolysis

Allele-specific oligonucleotide

Fluorophore-assisted carbohydrate elect

Amplicon

Frster resonance energy transfer

Function-spacer-lipid construct

Baby hamster kidney cell

Bio-layer interferometry

Gel doc

Branched DNA assay

Gene knockin

Gene knockout

GeneTalk

Calcium chloride transformation

Cell counting

3D cell culturing by magnetic levitation

Cell culture

Chemically defined medium

Chem-seq

ChIA-PET

ChIP-exo

ChIP-on-chip

ChIP-sequencing

Knockout moss

Chromatin immunoprecipitation

Kodecyte

Chromogenic in situ hybridization

Kodevirion

COLD-PCR

Combined bisulfite restriction analysis

Ligase chain reaction

Community Fingerprinting

Ligation (molecular biology)

Competition-ChIP

Helicase-dependent amplification

Immunoprecipitation

Isoelectric focusing

Isopeptag

Jumping library

Magnet-assisted transfection

DNA footprinting

MassTag-PCR

DNA microarray

MaxamGilbert sequencing

DNA Patterns

Methods to investigate proteinprotein in

DNA sequencing

Microsatellite enrichment

DNA Specimen Provenance Assignment

Minusheet perfusion culture system

DNase-Seq

Mutagenesis (molecular biology techniqu

Eastern blot

Northern blot

Exome sequencing

Northwestern blot

Extension Poly(A) Test

Nuclease protection assay

Nucleic acid structure determination

FAIRE-Seq

Far-Eastern blotting

Oligomer restriction

Far-western blotting

Overlap extension polymerase chain rea

Paired-end tag

PBLU

PBR322

1)Alanine scanning
In molecular biology, alanine scanning is a technique used to determine the contribution of a specific
residue to the stability or function of given protein. Alanine is used because of its non-bulky, chemically
inert, methyl functional group that nevertheless mimics the secondary structure preferences that many
of the other amino acids possess. Sometimes bulky amino acids such as valine or leucine are used in
cases where conservation of the size of mutated residues is needed.This technique can also be used to
determine whether the side chain of a specific residue plays a significant role in bioactivity. This is
usually accomplished by site-directed mutagenesis or randomly by creating a PCR library. Furthermore,
computational methods to estimate thermodynamic parameters based on a theoretical alanine
substitutions have been developed.This technique is rapid, because many side chains are analyzed
simultaneously and the need for protein purification and biophysical analysis is circumvented. The
technology is very mature at this point and is widely used in biochemical fields. The data can be tested
byIR, NMR Spectroscopy, mathematical methods, bioassays, etc. One good example of alanine
scanning is the examination of the role of charged residues on the surface of proteins. In a systematic
study on the roles of conserved charged residues on the surface of epithelial sodium channel (ENaC),
alanine scanning was used to reveal the importance of charged residues for the process of transport of
.the proteins to the cell surface

allele-specific oligonucleotide (2

An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary

to the sequence of a variable target DNA. It acts as a probe for the presence of the target
in a Southern blot assay or, more commonly, in the simpler Dot blot assay. It is a common
.tool used in genetic testing, forensics, and Molecular Biology research
An ASO is typically an oligonucleotide of 1521 nucleotide bases in length. It is designed
(and used) in a way that makes it specific for only one version, or allele, of the DNA being
tested. The length of the ASO, which strand it is chosen from, and the conditions by
which it is bound to (and washed from) the target DNA all play a role in its specificity.
These probes can usually be designed to detect a difference of as little as 1 base in the
target's genetic sequence, a basic ability in the assay of single-nucleotide
polymorphisms(SNPs), important in genotype analysis and the Human Genome Project.
To be detected after it has bound to its target, the ASO must be labeled with a
radioactive, enzymatic, or fluorescent tag. The Illumina Methylation Assay technology
takes advantage of ASO to detect one base pair difference (cytosine versus thymine) to
measure methylation at a specific CpG site

amplicon (3
An amplicon is a piece of DNA or RNA that is the source and/or product of natural or
artificial amplification or replication events. It can be formed using various methods
including polymerase chain reactions (PCR), ligase chain reactions (LCR), or
natural gene duplication. In this context, "amplification" refers to the production of one or
more copies of a genetic fragment or target sequence, specifically the amplicon. As the

product of an amplification reaction, amplicon is used interchangeably with common

.laboratory terms, such as PCR product
Artificial amplification is used in research, forensics, and medicinefor purposes that
include detection and quantification of infectious agents, identification of human
.remains, and extracting genotypes from human hair
Natural gene duplication is implicated in several forms of human cancer including primary
mediastinal B cell lymphoma and Hodgkin's lymphoma Amplicons in this context can
refer both to sections of chromosomal DNA that have been excised, amplified, and
reinserted elsewhere in the genome, and to extrachromasomal DNA known as double
minutes, each of which can be composed of one or more genes. Amplification of the
genes encoded by these amplicons generally increases transcription of those genes and
.ultimately the volume of associated proteins

4) Baby Hamster Kidney

Baby Hamster Kidney fibroblasts (aka BHK cells) are an adherent cell line used in molecular biology.
The cells were derived in 1961 by I. A. Macpherson and M. G. P. Stoker. Nowadays occasionally used,
subclone 13 which originally derived by single-cell isolation from the kidneys of five unsexed, 1-dayold hamsters.

5) Bio-layer interferometry
Bio-layer interferometry is a label-free technology for measuring biomolecular interactions
within the interactome. It is an optical analytical technique that analyzes
the interference pattern of white light reflected from two surfaces: a layer of
immobilized protein on the biosensor tip, and an internal reference layer Any change in
the number of molecules bound to the biosensor tip causes a shift in the interference
pattern that can be measured in real-time
The binding between a ligand immobilized on the biosensor tip surface and an analyte in
solution produces an increase in optical thickness at the biosensor tip, which results in

a wavelength shift, , which is a direct measure of the change in thickness of the

biological layer. Interactions are measured in real time, providing the ability to monitor
binding specificity, rates of association and dissociation, or concentration, with precision
.and accuracy
Only molecules binding to or dissociating from the biosensor can shift the interference
pattern and generate a response profile. Unbound molecules, changes in the refractive
index of the surrounding medium, or changes in flow rate do not affect the interference
pattern. This is a unique characteristic of bio-layer interferometry and extends its
capability to perform in crude samples used in applications for protein-protein
.interactions, quantitation, affinity, and kinetics
Bio-layer interferometry was pioneered by the founders of ForteBio, an instrument
.manufacturer with headquarters in Menlo Park California

branched DNA assay (6

In biology, a branched DNA assay is a signal amplification assay (as opposed to a target
.amplification assay) that is used to detect nucleic acid molecules
From the base up, a branched DNA assay begins with a dish or some other solid support
(e.g., a plastic dipstick). The dish is peppered with small, single stranded DNA molecules
(or chains) that 'stick up' into the air. These are known as capture probe DNA molecules.
Next, an extender DNA molecule is added. Each extender has two domains, one that
hybridizes to the capture DNA molecule and one that "hangs out" in the air. The purpose
of the extender is two-fold. First, it creates more available surface area for target DNA
molecules to bind, and second, it allows the assay to be easily adapted to detect a
.variety of target DNA molecules
Once the capture and extender molecules are in place and they have hybridized, the
sample can be added. Target molecules in the sample will bind to the extender molecule.

So we have a base peppered with capture probes, which are hybridized to extender
.probes, which in turn are hybridized to target molecules
At this point, signal amplification takes place. A label extender DNA molecule is added
that has two domains (similar to the first extender). The label extender hybridizes to the
target and to a pre-amplified molecule. The preamplifier molecule has two domains. First,
it binds to the label extender and second, it binds to the amplifier molecule. An example
amplifier molecule is an oligonucleotide chain bound to the enzyme alkaline
.phosphatase
Diagrammatically, we have Base -> Capture Probe -> Extender -> Target -> label
extender -> pre-amplifier -> amplifier
The assay can be used to detect and quantify many types of RNA or DNA target. In the
assay, branched DNA is mixed with a sample to be tested. The detection is done using a
non-radioactive method and does not require preamplification of the nucleic acid to be
detected. The assay entirely relies on hybridization. Enzymes are used to indicate the
extent of hybridization but are not used to manipulate the nucleic acids. Thus, small
amounts of a nucleic acid can be detected and quantified without a reverse
transcription step (in the case of RNA) and/or PCR. The assay can be run as a high
throughput assay, unlike quantitative Northern-blotting or the RNAse-protection assay,
which are labor-intensive and thus difficult to perform on a large number of samples. The
other major high throughput technique employed in the quantification of specific RNA
.molecules isquantitative PCR, after reverse transcription of the RNA to cDNA
Several different short single-stranded DNA molecules (oligonucleotides) are used in a
branched DNA-assay. The capture and capture-extender oligonucleotide bind to the
target nucleic acid and immobilize it on a solid support. The label oligonucleotide and the
branched DNA then detects the immobilized target nucleic acid. The immobilization of the
target on a solid support makes extensive washing easier, which reduces false positive
results. After binding of the target to the solid support it can be detected by branched
DNA which is coupled to an enzyme (e.g. alkaline phosphatase). The branched DNA
binds to the sample nucleic acid by specific hybridization in areas which are not occupied
by capture hybrids. The branching of the DNA allows for very dense decorating of the
DNA with the enzyme, which is important for the high sensitivity of the assay[The enzyme
catalyzes a reaction of a substrate which generates light (detectable in a luminometer).
The amount of light emitted increases with the amount of the specific nucleic acid present
in the sample. The design of the branched DNA and the way it is hybridized to the nucleic
acid to be investigated differs between different generations of the bDNA assay. Despite
the fact that the starting material is not preamplified, bDNA assays can detect less than
.100 copies of HIV-RNA per mL of blood

Calcium chloride (CaCl2) transformation (7

Calcium chloride (CaCl2) transformation is a laboratory technique
in prokaryotic (bacterial) cell biology. It increases the ability of a prokaryotic cell to
incorporate plasmid DNAallowing them to be genetically transformed.[1] The addition
of calcium chloride to a cell suspension promotes
the binding of plasmid DNA to lipopolysaccharides (LPS). Positively
charged calcium ions attract both the negatively charged DNA backbone and the
negatively charged groups in the LPS inner core. The plasmid DNA can then pass into
the cell upon heat shock, where chilled cells (+4 degrees Celsius) are heated to a higher
.temperature (+42 degrees Celsius) for a short time

Cell counting (8
Cell counting is a general name for various methods for the quantification of cells in life
.sciences, including medical diagnosis and treatment

3D cell culture by the magnetic levitation (9

3D cell culture by the magnetic levitation method (MLM) is the application of growing 3D
tissue by inducing cells treated with magnetic nanoparticle assemblies in spatially varying
magnetic fields using neodymium magnetic drivers and promoting cell to cell interactions
by levitating the cells up to the air/liquid interface of a standard petri dish. The magnetic
nanoparticle assemblies consist of magnetic iron oxide nanoparticles, gold nanoparticles,
and the polymer polylysine. 3D cell culturing is scalable, with the capability for culturing
500 cells to millions of cells or from single dish to high-throughput low volume
systemsOnce magnetized cultures are generated, they can also be use as the building
.block material, or the "ink", for the Magnetic 3D Bioprinting process

Cell culture (10

Cell culture is the process by which cells are grown under controlled conditions,
generally outside of their natural environment. In practice, the term "cell culture" now

refers to the culturing of cells derived from multi-cellular eukaryotes, especially animal
cells. However, there are also cultures of plants, fungi, insects and microbes,
including viruses, bacteria and protists. The historical development and methods of cell
.culture are closely interrelated to those of tissue culture and organ culture
The laboratory technique of maintaining live cell lines (a population of cells derived from
a single cell and containing the same genetic makeup) separated from their original
tissue source became more robust in the middle 20th century

chemically defined medium (11

A chemically defined medium is a growth medium suitable for the in vitro cell
culture of human or animal cells in which all of the chemical components are known. Standard
cell culture media are commonly supplemented with animal serum (such as fetal bovine
serum, FBS) as a source of nutrients and other ill-defined factors. The technical
disadvantages to using serum include its undefined nature, batch-to-batch variability in
.composition, and the risk of contamination
There is a clear distinction between serum-free media and chemically defined media. Serumfree media may contain undefined animal-derived products such as serum albumin (purified
from blood), hydrolysates, growth factors, hormones, carrier proteins, and attachment factors.
These undefined animal-derived products will contain complex contaminants, such as the lipid
content of albumin. In contrast, chemically defined media require that all of the components
must be identified and have their exact concentrations known. Therefore a chemically defined
medium must be entirely free of animal-derived components and cannot contain either fetal
bovine serum, bovine serum albumin orhuman serum albumin. To achieve this chemically
defined media is commonly supplemented with recombinant versions of albumin and growth
factors, usually derived from rice or E. coli, or synthetic chemical such as the
.polymer polyvinyl alcohol which can reproduce some of the functions of BSA/HSA
The constituents of a chemically defined media include: a basal media (such as DMEM, F12,
or RPMI 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants and
energy sources), which is supplemented with recombinant albumin, chemically defined lipids,

recombinant insulin and/or zinc, recombinant transferrin or iron,selenium and an

.antioxidant thiol such as 2-mercaptoethanol or 1-thioglycerol

chem -seq(12
Chem-seq is a technique that is used to map genome-wide interactions between
small molecules and their protein targets in the chromatin of eukaryotic cell nuclei.[1] The
method employs chemical affinity capture coupled with massively parallel DNA
sequencing to identify genomic sites where small molecules interact with their target
proteins or DNA. It was first described by Lars Anders et al. in the January, 2014 issue of
.""Nature Biotechnology

ChIA-PET (13
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a technique that
incorporates chromatin immunoprecipitation (ChIP)-based enrichment,chromatin proximity
ligation, Paired-End Tags, and High-throughput sequencing to determine de novo long.range chromatin interactions genome-wide (Fullwood & Yijun, 2009)
Genes can be regulated by regions far from the promoter such as regulatory elements,
insulators and boundary elements, and transcription-factor binding sites (TFBS). Uncovering
the interplay between regulatory regions and gene coding regions is essential for
understanding the mechanisms governing gene regulation in health and disease(Maston et
al., 2006). ChIA-PET can be used to identify unique, functional chromatin interactions
between distal and proximal regulatory transcription-factor binding sites and thepromoters of
.the genes they interact with
ChIA-PET can also be used to unravel the mechanisms of genome control during processes
such as cell differentiation, proliferation, and development. By creating ChIAPETinteractome maps for DNA-binding regulatory proteins and promoter regions, we can
.better identify unique targets for therapeutic intervention (Fullwood & Yijun, 2009)

ChIP-exo (14
ChIP-exo is a chromatin immunoprecipitation based method for mapping the locations at
which a protein of interest (transcription factor) binds to the genome. It is a modification
of the ChIP-seq protocol, improving the resolution of binding sites from hundreds of base
pairs to less than one base pair. It employs the use of exonucleases to degrade strands
of the protein-bound DNA in the 5'-3' direction to within a small number of nucleotides of
the protein binding site. The nucleotides of the exonuclease-treated ends are determined
using some combination of DNA sequencing,microarrays, and PCR. These sequences

are then mapped to the genome to identify the locations on the genome at which the
.protein binds

15) ChIP-on-chip
ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin
immunoprecipitation("ChIP") with DNA microarray ("chip"). Like regular ChIP, ChIP-on-chip is used to
investigate interactions between proteins and DNA in vivo. Specifically, it allows the identification of
the cistrome, sum of binding sites, for DNA-binding proteins on a genome-wide basis.[1] Whole-genome
analysis can be performed to determine the locations of binding sites for almost any protein of interest. [1] As
the name of the technique suggests, such proteins are generally those operating in the context of chromatin.
The most prominent representatives of this class are transcription factors, replication-related proteins,
like ORC, histones, their variants, and histone modifications. The goal of ChIP-on-chip is to locate protein
binding sites that may help identify functional elements in the genome. For example, in the case of a
transcription factor as a protein of interest, one can determine its transcription factor binding sites throughout
the genome. Other proteins allow the identification of promoter regions, enhancers, repressors and silencing
elements, insulators, boundary elements, and sequences that control DNA replication. [2] If histones are
subject of interest, it is believed that the distribution of modifications and their localizations may offer new
insights into the mechanisms of regulation. One of the long-term goals ChIP-on-chip was designed for is to
establish a catalogue of (selected) organisms that lists all protein-DNA interactions under various
physiological conditions. This knowledge would ultimately help in the understanding of the machinery behind

gene regulation, cell proliferation, and disease progression. Hence, ChIP-on-chip offers not only huge
potential to complement our knowledge about the orchestration of the genome on the nucleotide level, but
also on higher levels of information and regulation as it is propagated by research on epigenetics.

16) ChIP-sequencing
ChIP-sequencing, also known as ChIP-Seq or ChIP-seq, is a method used to analyze protein interactions
with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA
sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding
sites precisely for any protein of interest. Previously, ChIP-on-chip was the most common technique utilized
to study these proteinDNA relations

17) Chromatin Immunoprecipitation

Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation experimental
technique used to investigate the interaction between proteins and DNA in the cell. It aims to
determine whether specific proteins are associated with specific genomic regions, such
as transcription factors on promoters or other DNA binding sites, and possibly
defining cistromes. ChIP also aims to determine the specific location in the genome that
various histone modifications are associated with, indicating the target of the histone
modifiers.[1]
Briefly, the method is as follows: protein and associated chromatin in living cells or tissues are
temporarily bonded, the DNA-protein complexes (chromatin-protein) are then sheared into
~500 bp DNA fragments by sonication, cross-linked DNA fragments associated with the
protein(s) of interest are selectively immunoprecipitated from the cell debris using appropriate
protein-specific antibody, the associated DNA fragments are purified and their sequence is
determined. These DNA sequences are supposed to be associated with the protein of
interest in vivo

18) Chromogenic in situ hybridization (CISH),

Chromogenic in situ hybridization (CISH), is a process in which a labeled
complementary DNA or RNA strand is used to localize a specific DNA or RNA sequence
in a tissue specimen. CISH methodology may be used to evaluate gene
amplification, gene deletion, chromosome translocation, and chromosome number. CISH
uses conventional peroxidase or alkaline phosphatase reactions visualized under a
standard bright-field microscope, and is applicable to formalin-fixed, paraffin-embedded

(FFPE) tissues, blood or bone marrow smears, metaphase chromosome spreads, and
.fixed cells.CISH is an alternative to fluorescent in situ hybridization

COLD-PCR (19
COLD-PCR (co-amplification at lower denaturation temperature-PCR) is a
modified Polymerase Chain Reaction (PCR) protocol that enriches variant alleles from a
mixture of wildtype and mutation-containing DNA. The ability to preferentially amplify and
identify minority alleles and low-level somatic DNA mutations in the presence of excess
wildtype alleles is useful for the detection of mutations. Detection of mutations is
important in the case of early cancer detection from tissue biopsies and body fluids such
as blood plasmaor serum, assessment of
residual disease after surgery or chemotherapy, disease staging and molecular profiling
for prognosis or tailoring therapy to individual patients, and monitoring of therapy
outcome and cancer remission or relapse. Common PCR will amplify both the major
(wildtype) and minor (mutant) alleles with the same efficiency, occluding the ability to
easily detect the presence of low-level mutations. The capacity to detect a mutation in a
mixture of variant/wildtype DNA is valuable because this mixture of variant DNAs can
occur when provided with a heterogeneous sample as is often the case with cancer
biopsies. Currently, traditional PCR is used in tandem with a number of different
downstream assays for genotyping or the detection of somatic mutations. These can
include the use of amplified DNA for RFLP analysis, MALDI-TOF (matrix-assisted laserdesorptiontime-of-flight) genotyping, or direct sequencing for detection of mutations
by Sanger sequencing or pyrosequencing. Replacing traditional PCR with COLD-PCR for
these downstream assays will increase the reliability in detecting mutations from mixed
samples, including tumors and body fluids

Combined Bisulfite Restriction Analysis (20

Combined Bisulfite Restriction Analysis (or COBRA) is a molecular biology technique that
allows for the sensitive quantification of DNA methylation levels at a specific genomic
locus on a DNA sequence in a small sample of genomic DNA. The technique is a
variation of bisulfite sequencing, and combines bisulfite conversion based polymerase
chain reaction with restriction digestion. Originally developed to reliably handle minute
amounts of genomic DNA from microdissected paraffin-embedded tissue samples, the
technique has since seen widespread usage in cancer researchand epigenetics studies

Community fingerprinting (21

Community fingerprinting refers to a set of molecular biology techniques that can be used
to quickly profile the diversity of a microbial community. Rather than directly identifying or
counting individual cells in an environmental sample, these techniques show how many
variants of a gene are present. In general, it is assumed that each different gene variant
represents a different type of microbe. Community fingerprinting is used
by microbiologists studying a variety of microbial systems (e.g. marine, freshwater, soil,
and human microbial communities) to measure biodiversity or track changes in
community structure over time. The method analyzes environmental samples by
assaying genomicDNA. This approach offers an alternative to microbial culturing, which
is important because most microbes cannot be cultured in the laboratory.[1] Community
fingerprinting does not result in identification of individual microbe species; instead, it
.presents an overall picture of a microbial community

Competition-ChIP (22
Competition-ChIP is variant of the Chip-Sequencing protocol, used to measure relative
binding dynamics of a transcription factor (TF) on DNA. Since TF occupancy measures
are thought to be a poor predictor of TF function at a given locus, Competition-ChIP is
much more strongly linked to function than occupancy

DNA footprinting (23

DNA footprinting is a method of investigating the sequence specificity of DNAbinding proteins in vitro. This technique can be used to study protein-DNA interactions
.both outside and within cells

The regulation of transcription has been studied extensively, and yet there is still much
that is not known. Transcription factors and associated proteins that
bind promoters, enhancers, or silencers to drive or repress transcription are fundamental
to understanding the unique regulation of individual genes within the genome.
Techniques like DNA footprinting will help elucidate which proteins bind to these regions
.of DNA and unravel the complexities of transcriptional control

DNA microarray (24

A DNA microarray (also commonly known as DNA chip or biochip) is a collection of
microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to
measure the expression levels of large numbers of genes simultaneously or
to genotype multiple regions of a genome. Each DNA spot
contains picomoles (1012 moles) of a specific DNA sequence, known
as probes (or reporters or oligos). These can be a short section of a gene or other DNA
element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample
(called target) under high-stringency conditions. Probe-target hybridization is usually
detected and quantified by detection of fluorophore-, silver-, or chemiluminescence.labeled targets to determine relative abundance of nucleic acid sequences in the target

DNA patterns (25

DNA patterns are graphs of DNA or RNA sequences. Various functional structures such
as promoters and genes, or larger structures like bacterial or viral genomes, can be
analyzed using DNA patterns

DNA sequencing (26

DNA sequencing is the process of determining the precise order of nucleotides within
a DNA molecule. It includes any method or technology that is used to determine the
order of the four basesadenine, guanine, cytosine, and thyminein a strand of DNA.
The advent of rapid DNA sequencing methods has greatly accelerated biological and
medical research and discovery.
Knowledge of DNA sequences has become indispensable for basic biological research,
and in numerous applied fields such as diagnostic, biotechnology, forensic
biology, virology and biological systematics. The rapid speed of sequencing attained with
modern DNA sequencing technology has been instrumental in the sequencing of
complete DNA sequences, or genomes of numerous types and species of life, including
the human genome and other complete DNA sequences of many animal, plant,
and microbial species.

An example of the results of automated chain-termination DNA sequencing.

The first DNA sequences were obtained in the early 1970s by academic researchers
using laborious methods based on two-dimensional chromatography. Following the
development of fluorescence-based sequencing methods with automated analysis,[1]DNA
sequencing has become easier and orders of magnitude faster

27) DNA Specimen Provenance Assignment (DSPA)

DNA Specimen Provenance Assignment (DSPA) is a molecular diagnostic test used to
definitively assign biopsy specimen identity during the diagnostic testing cycle
for cancerand other histopathological conditions. The term first appeared in the 2011
scientific paper, The Changing Spectrum of DNA-Based Specimen Provenance Testing
in Surgical Pathology, published in the American Journal of Clinical Pathology which
built upon concepts described in an earlier paper published in the Journal of Urology

28) DNase-Seq
DNase-Seq (DNase I hypersensitive sites sequencing) is a method in molecular
biology used to identify the location of regulatory regions, based on the genome-wide
sequencing of regions super sensitive to cleavage by DNase I.[1][2] FAIRE-Seq is a
successor of DNase-Seq for the genome-wide identification of accessible DNA regions in
the genome. Both the protocols for determining open chromatin region have their own
bias depending on underlying nucleosome structure. Such as FAIRE-seq provides higher
tag-count at non-promoter regions.[3] On the other hand, Dnase-seq signal is higher at
promoter regions, however DNase-seq has been shown to have better sensitivity than
FAIRE-seq even at non-promoter regions

29) eastern blot

The eastern blot is a biochemical technique used to analyze protein post translational
modifications (PTM) such as lipids, phosphomoieties and glycoconjugates. It is most
often used to detect carbohydrate epitopes. Thus, Eastern blotting can be considered an
extension of the biochemical technique of Western blotting. Multiple techniques have
been described by the term Eastern blotting, most use proteins blotted from SDSPAGE gel on to a PVDF or nitrocellulose membrane. Transferred proteins are analyzed
for post-translational modifications using probes that may
detect lipids, carbohydrate, phosphorylation or any other protein modification. Eastern
blotting should be used to refer to methods that detect their targets through specific
interaction of the PTM and the probe, distinguishing them from a standard Far-western
blot. In principle, Eastern blotting is similar to lectin blotting (i.e. detection of carbohydrate
epitopes on proteins or lipids

30) Exome sequencing

Exome sequencing is a technique for sequencing all the protein-coding genes in a
genome (known as the exome). It consists of first selecting only the subset of DNA that
encodes proteins (known as exons), and then sequencing that DNA using any high
throughput DNA sequencing technology. There are 180,000 exons, which constitute
about 1% of the human genome, or approximately 30 million base pairs, but mutations in
these sequences are much more likely to have severe consequences than in the
remaining 99%.[1] The goal of this approach is to identify genetic variation that is
responsible for both mendelian and common diseases such as Miller
syndrome andAlzheimer's disease without the high costs associated with whole-genome
sequencing.

31) extension Poly(A) Test (ePAT)

The extension Poly(A) Test (ePAT) describes a method to determine the poly(A) tail
.lengths of mRNA molecules. It was developed and described by A. Jnicke et al. in 2012
The method consists of three separate steps: In the first step, the poly-adenylated RNA is
hybridised to a DNA oligonucleotide featuring a poly-deoxythymidine sequence at its 5
end. Klenow polymerase then catalyses elongation of the mRNAs 3 end, using the DNA
oligonucleotide as a template. This reaction takes place at 25 C. In the second step,
reverse transcriptase synthesises extends the DNA oligonucleotides that have annealed
to the mRNAs extended 3 end. In order to ensure that DNA oligomers hybridised to
internal poly(A) sequences do not serve as primers for reverse transcription, the second
step is carried out at 55 C. A third and final step involves amplification of the newly
synthesised cDNA via PCR. This PCR requires one gene-specific and one universal
primer. Analysis of the amplicons lengths allows for estimation of the sequence flanked
.by the two primers, i.e. the poly(A) tail length of the sample mRNA

FAIRE-Seq (32
FAIRE-Seq (Formaldehyde-Assisted Isolation of Regulatory Elements) is a method
in molecular biology used for determining the sequences of those DNA regions in
the genomeassociated with regulatory activity.[1] The technique was developed in the
laboratory of Jason D. Lieb at the University of North Carolina, Chapel Hill. In contrast
to DNase-Seq, the FAIRE-Seq protocol doesn't require the permeabilization of cells or
isolation of nuclei, and can analyse any cell types. In a study of seven diverse human cell
types, DNase-seq and FAIRE-seq produced strong cross-validation, with each cell type
.having 1-2% of the human genome as open chromatin
The protocol is based on the fact that the formaldehyde cross-linking is more efficient
in nucleosome-bound DNA than it is in nucleosome-depleted regions of the genome. This
method then segregates the non cross-linked DNA that is usually found in open
chromatin, which is then sequenced. The protocol consists of cross linking, phenol
.extraction and sequencing the DNA in aqueous phase

Far-Eastern blotting (33

Far-Eastern blotting is a technique developed in 1994 by Taki and colleagues[1] at the
Tokyo Medical and Dental University, Japan for the analysis of lipids separated by highperformance thin layer chromatography (HPTLC). The lipids are transferred from the
HPTLC plate to a PVDF membrane for further analysis, for example by enzymatic or
ligand binding assays[1] or mass spectrometry.[2]
Cholesterol, glycerophospholipids and sphingolipids are major constituents of the cell
membrane and in certain cases function as second messengers in cell
proliferation,apoptosis and cell adhesion in inflammation and tumor metastasis. Fareastern blotting was established as a method for transferring lipids from an HPTLC plate
to a polyvinyledene difluoride (PVDF) membrane within a minute. Applications of this with
other methods have been studied. Far-eastern blotting allows for the following
techniques:

Purification of glycosphingolipids and phospholipids.

Structural analysis of lipids in conjunction with direct mass spectrometry.

Binding study using various ligands such as antibodies, lectins, bacterium,

viruses, and toxins, and

Enzyme reaction on membranes.

Not only analysis of lipids but also metabolites of drugs and natural compounds from
plants, and environmental hormones are possible by this method.

34) Far-western blotting

Far-western blotting is a molecular biological method which is based on the technique
of western blotting to detect protein-protein interaction in vitro. While usual western
blotting uses an antibody to detect a protein of interest, far-western blotting uses a nonantibody protein, which can bind the protein of interest. Thus, whereas western blotting is
used for the detection of certain proteins, far-western blotting is rather employed to detect
protein:protein interactions.

35) Fast parallel proteolysis

Fast parallel proteolysis (FASTpp) is a method to determine
the thermostability of proteins that does not need previous purification and labeling

36) Fluorophore assisted carbohydrate electrophoresis

Fluorophore assisted carbohydrate electrophoresis or FACE is
a biochemical technology suited for detecting complex mixtures of high molecular
weight N-glycans. A specialized form of this technique is the DSA-FACE, which is an
acronym for DNA sequencer-assisted flurophore-assisted carbohydrate electrophoresis.
DSA-FACE has higher resolution and sensitivity than classical FACE

37) Frster resonance energy transfer

Frster resonance energy transfer (FRET), fluorescence resonance energy
transfer (FRET), resonance energy transfer (RET) orelectronic energy transfer (EET) is a
mechanism describing energy transfer between two light-sensitive molecules
(chromophores).[1] A donor chromophore, initially in its electronic excited state, may
transfer energy to an acceptor chromophore through nonradiative dipoledipole
coupling. The efficiency of this energy transfer is inversely proportional to the sixth power
of the distance between donor and acceptor, making FRET extremely sensitive to small
changes in distance
Measurements of FRET efficiency can be used to determine if two fluorophores are
within a certain distance of each other. Such measurements are used as a research tool
.in fields including biology and chemistry
FRET is analogous to near-field communication, in that the radius of interaction is much
smaller than the wavelength of light emitted. In the near-field region, the excited
chromophore emits a virtual photon that is instantly absorbed by a receiving
chromophore. These virtual photons are undetectable, since their existence violates the
conservation of energy and momentum, and hence FRET is known as
aradiationless mechanism. Quantum electrodynamical calculations have been used to
determine that radiationless (FRET) and radiative energy transfer are the short- and longrange asymptotes of a single unified mechanism

Function-spacer-lipid constructs (38

Function-spacer-lipid constructs (FSL constructs) are amphiphatic,
water dispersible biosurface engineering constructs that can be used to engineer the
surface of cells, viruses and organisms, or to modify solutions and non-biological
surfaces with bioactives. FSL constructs spontaneously and stably incorporate into cell
membranes. FSL constructs with all these afore mentioned features are also known as
KODE constructs. The process of modifying surfaces with FSL constructs is known as
"koding" and the resultant "koded" cells, viruses and liposomes are respectively known
.as kodecytes kodevirions and kodesomes

gel doc (39

A gel doc, also known as a gel documentation system, gel image system or gel imager, is
equipment widely used in molecular biology laboratories for the imaging and
documentation of nucleic acid and protein suspended
within polyacrylamide or agarose gels. These gels are typically stained with ethidium
bromide or other fluorophores such asSYBR Green. Generally, a gel doc includes
an ultraviolet (UV) light transilluminator, a hood or a darkroom to shield external light
sources and protect the user from UV exposure, and a CCD camera for image capturing.
[1]

The main manufacturers of Gel documentation systems are Vilber Lourmat, Bioolympics
.and Biorad
Recently produced imager models also include features to handle a variety
.of fluorescence and chemiluminescence with cameras cooled to -67 C

Gene knock-in (40

In molecular cloning and biology, a Knock-in (or Gene knock-in) refers to a genetic
engineering method that involves the insertion of a protein coding cDNA sequence at a
particular locus in an organism's chromosome.[1] Typically, this is done in mice since the
technology for this process is more refined, and because mouse embryonic stem cells
are easily manipulated. The difference between knock-in technology
and transgenic technology is that a knock-in involves a gene inserted into a specific
.locus, and is a "targeted" insertion
A common use of knock-in technology is for the creation of disease models. It is a
technique by which scientific investigators may study the function of the regulatory
machinery (e.g. promoters) that governs the expression of the natural gene being
replaced. This is accomplished by observing the new phenotype of the organism in
question. The BACsand YACs are used in this case so that large fragments can be
.transferred

gene knockout (41

A gene knockout (abbreviation: KO) is a genetic technique in which one of
an organism's genes are made inoperative ("knocked out" of the organism). Also known
asknockout organisms or simply knockouts, they are used in learning about a gene that
has been sequenced, but which has an unknown or incompletely known function.
Researchers draw inferences from the difference between the knockout organism and
.normal individuals
The term also refers to the process of creating such an organism, as in "knocking out" a
gene. The technique is essentially the opposite of a gene knockin. Knocking out two

genes simultaneously in an organism is known as a double knockout (DKO). Similarly the

terms triple knockout (TKO) and quadruple knockouts (QKO) are used to describe three
.or four knocked out genes, respectively

GeneTalk (42
GeneTalk is a web-based platform, tool, and database, for filtering, reduction and
prioritization of human sequence variants from next-generation sequencing (NGS) data.
GeneTalk allows editing annotation about sequence variants and build up a crowd
sourced database with clinically relevant information for diagnostics of genetic disorders.
GeneTalk allows searching for information about specific sequence variants and connects to

.experts on variants that are potentially disease-relevant

Helicase-dependent amplification (43

Helicase-dependent amplification (HDA) is a method for in vitro DNA amplification like
.the polymerase chain reaction (PCR), but that works at constant temperature

Immunoprecipitation (44
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution
using an antibody that specifically binds to that particular protein. This process can be
used to isolate and concentrate a particular protein from a sample containing many
thousands of different proteins. Immunoprecipitation requires that the antibody be
.coupled to a solid substrate at some point in the procedure

Isoelectric focusing (45

Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating
different molecules by differences in their isoelectric point (pI). It is a type of
zone electrophoresis, usually performed on proteins in a gel, that takes advantage of the
fact that overall charge on the molecule of interest is a function of the pH of its
surroundings

Isopeptag (46
Isopeptag is a 16 amino acid peptide tag that can be genetically linked to proteins without
interfering with protein folding.[1] What makes the isopeptag different from other peptide
tags is that it can bind its binding protein through a permanent and irreversible covalent
bond. Other peptide tags generally bind their targets through weak non-covalent
interactions, thus limiting their use in applications where molecules experience extreme
forces. The isopeptags covalent binding to its target overcomes these barriers and allows
..target proteins to be studied in harsher molecular environments

Jumping libraries (47

Jumping libraries or junction-fragment libraries are collections of genomic DNA fragments
generated by chromosome jumping. These libraries allow us to analyze large areas of
the genome and overcome distance limitations in common cloning techniques. A jumping
library clone is composed of two stretches of DNA that are usually located many
kilobases away from each other. The stretch of DNA located between these two ends is
deleted by a series of biochemical manipulations carried out at the start of this cloning
technique

knockout moss (48

A knockout moss is a moss plant in which one or more specific genes are deleted or
inactivated ("knocked out") by gene targeting. After deletion of a gene, the knockout
moss has lost the trait encoded by this gene. Thus, the function of this gene can be
inferred. This scientific approach is called reverse genetics as the scientist wants to
unravel the function of a specific gene. In classical genetics the scientist starts with
a phenotype of interest and searches for the gene that causes this phenotype. Knockout
.mosses are relevant for basic research in biology as well as in biotechnology

kodecyte (49
A kodecyte is a living cell that has been modified (koded) by the incorporation of one or
more function-spacer-lipid constructs(FSL constructs)[1][2] to gain a new or novel biological,
chemical or technological function. The cell is modified by the lipid tail of the FSL
.construct incorporating into the bilipid membrane of the cell
All kodecytes retain their normal vitality and functionality while gaining the new function of
the inserted FSL constructs. The combination of dispersibility in biocompatible media,
spontaneous incorporation into cell membranes, and apparent low toxicity, makes FSL
constructs suitable as research tools and for the development of new diagnostic
and therapeutic applications

ligase chain reaction (50

The ligase chain reaction (LCR) is a method of DNA amplification. While the betterknown PCR carries out the amplification by polymerizing nucleotides, LCR instead
amplifies the nucleic acid used as the probe. For each of the two DNA strands, two partial
probes are ligated to form the actual one; thus, LCR uses two enzymes: a DNA
polymerase and a DNA ligase. Each cycle results in a doubling of the target nucleic acid
.molecule. A key advantage of LCR is greater specificity as compared to PCR

Ligation (51
Ligation in molecular biology is the joining of two nucleic acid fragments through the
action of an enzyme. It is an essential laboratory procedure in the molecular cloning of
DNA whereby DNA fragments are joined together to create recombinant DNA molecules,
such as when a foreign DNA fragment is inserted into a plasmid. The ends of DNA
fragments are joined together by the formation of phosphodiester bonds between the 3'hydroxyl of one DNA terminus with the 5'-phosphoryl of another. RNA may also be ligated
.+similarly. A co-factor is generally involved in the reaction, and this is usually ATP or NAD
Ligation in the laboratory is normally performed using T4 DNA ligase, however,
procedures for ligation without the use of standard DNA ligase are also popular

Magnet Assisted Transfection (52

Magnet Assisted Transfection is a transfection method, which uses magnetic force to
deliver DNA into target cells. Therefore, nucleic acids are first associated with magnetic
nanoparticles. Then, application of magnetic force drives the nucleic acid-parcticle
complexes towards and into the target cells, where the cargo is release

MaxamGilbert sequencing (53

MaxamGilbert sequencing is a method of DNA sequencing developed by Allan

Maxam and Walter Gilbert in 19761977. This method is based on nucleobase-specific
partial chemical modification of DNA and subsequent cleavage of the DNA backbone at
sites adjacent to the modified nucleotides.[1]

An example MaxamGilbert sequencing reaction. Cleaving the same tagged segment of DNA at
different points yields tagged fragments of different sizes. The fragments may then be separated
by gel electrophoresis.

MaxamGilbert sequencing was the first widely adopted method for DNA sequencing,
and, along with the Sanger dideoxy method, represents the first generation of DNA
sequencing methods. MaxamGilbert sequencing is no longer in widespread use, having
been supplanted by next-generation sequencing methods.

54) proteinprotein interactions.

There are many methods to investigate proteinprotein interactions. Each of the
approaches has its own strengths and weaknesses, especially with regard to
the sensitivity and specificity of the method. A high sensitivity means that many of the
interactions that occur in reality are detected by the screen. A high specificity indicates
that most of the interactions detected by the screen are occurring in reality

55) Microsatellite enrichment

Microsatellite enrichment is a method in molecular biology used for enriching the amount
of microsatellite sequences in a DNA sample. This can be achieved by
designingoligonucleotide probes that hybridize with the repeats in the microsatellites and
then pull out the probe/microsatellite complexes from the solution. [1] This has been shown
to be a cost-effective method to sample the genetic diversity in non-model organisms
.

Minusheet perfusion culture system (56

Minusheet perfusion culture system is used for advanced cell culture experiments in
combination with adherent cells and to generate specialized tissues in combination with
.selected biomaterials, special tissue carriers and compatible perfusion culture containers
The technical development of the Minusheet perfusion culture system was driven by the
idea to create under in vitro conditions an environment resembling as near as possible
the situation of specialized tissues found within the organism. Basis of this invention is
therefore individually selected biomaterials for optimal cell adhesion mounted in
Minusheet tissue carriers. Moreover, to always offer fresh nutrition including respiratory
gas and to simulate a tissue-specific fluid environment, the tissue carriers can be inserted
into compatible perfusion culture containers. As a result, a variety of publications
illustrates that tissues generated by this innovative approach exhibit an excellent and
stable quality. Thus, on the one hand the system provides a highly adaptable basis for
the culture of adherent cells and the generation of specialized tissues. On the other hand
the Minusheet perfusion culture system is bridging a methodical gap between the
conventional static 24 well culture plate and modern perfusion culture technology

Mutagenesis (57
Mutagenesis in the laboratory is an important technique whereby DNA mutations are
deliberately engineered to produce mutant genes, proteins, strains of bacteria, or
othergenetically-modified organisms. Various constituents of a gene, such as its control
elements and its gene product, may be mutated so that the functioning of a gene or
protein can be examined in detail. The mutation may also produce mutant proteins with
interesting properties, or enhanced or novel functions that may be of commercial use.
Mutants strains may also be produced that have practical application or allow the
.molecular basis of particular cell function to be investigated

northern blot (58

the northern blot is a technique used in molecular biology research to study gene
expression by detection of RNA (or isolated mRNA) in a sample.

Flow diagram outlining the general procedure for RNA detection by northern blotting.

With northern blotting it is possible to observe cellular control over structure and function
by determining the particular gene expression levels during
differentiation, morphogenesis, as well as abnormal or diseased conditions.[3] Northern
blotting involves the use ofelectrophoresis to separate RNA samples by size and
detection with a hybridization probe complementary to part of or the entire target
sequence. The term 'northern blot' actually refers specifically to the capillary transfer of
RNA from the electrophoresis gel to the blotting membrane. However, the entire process
is commonly referred to as northern blotting.[4] The northern blot technique was developed
in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.
[5]

Northern blotting takes its name from its similarity to the first blotting technique,

the Southern blot, named for biologist Edwin Southern.[1] The major difference is that
RNA, rather than DNA, is analyzed in the northern blot

59) Northwestern blot

The Northwestern blot, also known as the Northwestern assay, is a hybrid analytical
technique of the Western blot and the Northern blot, and is used in molecular biology to
detect interactions between RNA and proteins. A related technique, the Western blot, is
used to detect a protein of interest that involves transferring proteins that are separated
by gel electrophoresis onto a nitrocellulose membrane. A colored precipitate clusters
along the band on the membrane containing a particular target protein. A Northern blot is
a similar analytical technique that, instead of detecting a protein of interest, is used to
study gene expression by detection of RNA (or isolated mRNA) on a similar membrane.
The Northwestern blot combines the two techniques, and specifically involves the
identification of labeled RNA that interact with proteins that are immobilized on a similar
nitrocellulose membrane.

60) Nuclease protection assay

Nuclease protection assay is a laboratory technique used in biochemistry and genetics to
identify individual RNA molecules in a heterogeneous RNA sample extracted fromcells.
The technique can identify one or more RNA molecules of known sequence even at low
total concentration. The extracted RNA is first mixed with antisense RNA or DNAprobes
that are complementary to the sequence or sequences of interest and the
complementary strands are hybridized to form double-stranded RNA (or a DNA-RNA
hybrid). The mixture is then exposed to ribonucleases that specifically cleave only singlestranded RNA but have no activity against double-stranded RNA. When the reaction runs
to completion, susceptible RNA regions are degraded to very short oligomers or to
individual nucleotides; the surviving RNA fragments are those that were complementary
to the added antisense strand and thus contained the sequence of interest.

61) Structure probing of nucleic acids

Structure probing of nucleic acids is the process by which biochemical techniques are
used to determine nucleic acid structure.[1] This analysis can be used to define the
patterns which can infer the molecular structure, experimental analysis of molecular
structure and function, and further understanding on development of smaller molecules
for further biological research.[2] Structure probing analysis can be done through many
different methods, which include chemical probing, hydroxyl radical probing, SHAPE,
nucleotide analog interference mapping (NAIM), and in-line probing.

62) Oligomer Restriction

Oligomer Restriction (abbreviated OR) is a procedure to detect an altered DNA sequence
in a genome. A labeled oligonucleotide probe is hybridized to a target DNA, and then
treated with a restriction enzyme. If the probe exactly matches the target, the restriction
enzyme will cleave the probe, changing its size. If, however, the target DNA does not
exactly match the probe, the restriction enzyme will have no effect on the length of the

probe. The OR technique, now rarely performed, was closely associated with the
development of the popular polymerase chain reaction (PCR) method.

63) overlap extension polymerase chain reaction

The overlap extension polymerase chain reaction (or OE-PCR) is a variant of PCR. It is
also referred to as Splicing by overlap extension / Splicing by overhang extension (SOE)
PCR. It is used to insert specific mutations at specific points in a sequence or to splice
smaller DNA fragments into a larger polynucleotide.

64) Paired-end tags

Paired-end tags (PET) (sometimes "Paired-End diTags", or simply "ditags") are the short
sequences at the 5 and 3 ends of a DNA fragment which are unique enough that they
(theoretically) exist together only once in a genome, therefore making the sequence of the
DNA in between them available upon search (if full-genome sequence data is available) or
upon further sequencing (since tag sites are unique enough to serve as primer annealing
sites). Paired-end tags (PET) exist in PET libraries with the intervening DNA absent, that is, a
PET "represents" a larger fragment of genomic or cDNA by consisting of a short 5' linker
sequence, a short 5' sequence tag, a short 3' sequence tag, and a short 3' linker sequence. It

was shown conceptually that 13 base pairs are sufficient to map tags uniquely.[1] However,
longer sequences are more practical for mapping reads uniquely.
The endonucleases (discussed below) used to produce PETs give longer tags (18/20 base
pairs and 25/27 base pairs) but sequences of 50100 base pairs would be optimal for both
mapping and cost efficiency.[1] After extracting the PETs from many DNA fragments, they are
linked (concatenated) together for efficient sequencing. On average, 2030 tags could be
sequenced with the Sanger method, which has a longer read length.[1] Since the tag
sequences are short, individual PETs are well suited for next-generation sequencing that has
short read lengths and higher throughput. The main advantages of PET sequencing are its
reduced cost by sequencing only short fragments, detection of structural variants in the
genome, and increased specificity when aligning back to the genome compared to single
tags, which involves only one end of the DNA

65) pBLU
pBLU is a commercially produced bacterial plasmid that contains genes
for ampicillin resistance (specifically, beta lactamase) and beta galactosidase. It is often
used in conjunction with an ampicillin-susceptible E. coli strain to teach about
transformation of eubacteria. It is 5,437 base pairs long. There is a multiple cloning site in
the lacZ gene.

66) pBR322
pBR322 is a plasmid and was one of the first widely used E. coli cloning vectors. Created in
1977 in the laboratory of Herbert Boyer at theUniversity of California, Davis, it was named
after the Mexican postdoctoral researchers who constructed it. The p stands for "plasmid,"
".and BR for "Bolivar" and "Rodriguez
pBR322 is 4361 base pairs

[1]

in length and contains the replicon of plasmid pMB1,

the ampR gene, encoding the ampicillin resistanceprotein (source plasmid RSF2124) and
the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101). The
plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40
sites lie within the tetR gene. There are 2 sites for restriction enzymes HindIII and ClaI within
the promoter of the tetR gene. There are 6 key restriction sites inside the ampR gene. The
origin of replication or ori site in this plasmid is pMB1 (a close relative of ColE1). [2]
The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the
count increases through the tet gene. The ampicillin resistance gene is penicillin betalactamase. Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter,
and P1 is artificially created by the ligation of two different DNA fragments to create pBR322.
P2 is in the same region as P1, but it is on the opposite strand and initiates transcription in the
direction of the tetracycline resistance gene

Photoaffinity labeling (67

Photoaffinity labeling is a technique used to attach "labels" to the active site of a large
molecule, especially a protein. The "label" attaches to the molecule loosely and
reversibly, and has an inactive site which can be converted using photolysis into a highly
reactive form, which causes the label to bind more permanently to the large molecule. [1]
[2]

The technique was first described in the 1970s.[3] Molecules that have been used as

labels in this process are often analogs of complex molecules, in which certain functional
groups are replaced with an azide grou

Plant transformation vectors (68

Plant transformation vectors are plasmids that have been specifically designed to
facilitate the generation of transgenic plants. The most commonly used plant
transformation vectors are termed binary vectors because of their ability to replicate
in both E. coli, a common lab bacterium, and Agrobacterium tumefaciens, a
bacterium used to insert the recombinant (customized) DNA into plants. Plant
Transformation vectors contain three key elements;

Plasmids Selection (creating a custom circular strand of DNA)

Plasmids Replication (so that it can be easily worked with)

Transfer DNA (T-DNA) region (inserting the DNA into the agrobacteria)

69) plasmid
A plasmid is a small DNA molecule within a cell that is physically separated from
a chromosomal DNA and can replicate independently. They are most commonly found
in bacteria as small, circular, double-stranded DNA molecules; however, plasmids are
sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry

genes that may benefit the survival of the organism, for example antibiotic resistance. While
the chromosomes are big and contain all the essential information for living (an adequate
analogy is the hard-drive of a computer), plasmids usually are very small and contain
additional information (in this analogy, plasmids are the USB flash drives). Artificial plasmids
are widely used as vectors inmolecular cloning, serving to drive the replication of recombinant
.DNA sequences within host organisms
Plasmids are considered replicons, a unit of DNA capable of replicating autonomously within
a suitable host. However, plasmids, like viruses, are not considered by some to be a form
of life.[1] Plasmid can be transmitted from one bacterium to another (even of another species)
via three main mechanisms: transformation, transduction, and conjugation. This host-to-host
transfer of genetic material is called horizontal gene transfer, and plasmids can be considered
part of the mobilome. Unlike viruses (which encase their genetic material in a protective
protein coat called a capsid), plasmids are "naked" DNA and do not encode genes necessary
to encase the genetic material for transfer to a new host. However, some classes of plasmids
encode the conjugative "sex" pilus necessary for their own transfer. The size of the plasmid
varies from 1 to over 1,000 kbp,[2] and the number of identical plasmids in a single cell can
.range anywhere from one to thousands under some circumstances
The relationship between microbes and plasmid DNA is neither parasitic nor mutualistic,
because each implies the presence of an independent species living in a detrimental or
commensal state with the host organism. Rather, plasmids provide a mechanism for
horizontal gene transfer within a population of microbes and typically provide a selective
advantage under a given environmental state. Plasmids may carry genes that
provide resistance to naturally occurring antibiotics in a competitive environmental niche, or
the proteins produced may act as toxins under similar circumstances, or allow the organism to
utilize particular organic compounds that would be advantageous when nutrients are scarce

Plasmidome (70
The term Plasmidome refers to the total plasmids content that is available in a certain
environment.[1] The term is a portmanteau of the two English words Plasmid and
Kingdom. In biological research, plasmidome may refer to the actual plasmids that were
found and isolated from a certain microorganism by means of culturing isolated

microorganism and investigating the plasmids it possesses or by taking an environmental

sample and performing a metagenomic survey using next generation
sequencing methods in order to reveal and characterize plasmid genomes that belong to
that environment

polymerase chain reaction (PCR) (71

The polymerase chain reaction (PCR) is a technology in molecular biology used
to amplify a single copy or a few copies of a piece of DNA across several orders of
.magnitude, generating thousands to millions of copies of a particular DNA sequence
Developed in 1983 by Kary Mullis,[1][2] PCR is now a common and often indispensable
technique used in medical and biological research labs for a variety of applications. [3]
[4]

These include DNA cloning for sequencing, DNA-based phylogeny, or functional

analysis of genes; the diagnosis of hereditary diseases; the identification of genetic

fingerprints (used in forensic sciences and paternity testing); and the detection and
diagnosis of infectious diseases. In 1993, Mullis was awarded theNobel Prize in
Chemistry along with Michael Smith for his work on PCR.[5]
The method relies on thermal cycling, consisting of cycles of repeated heating and
cooling of the reaction for DNA meltingand enzymatic replication of the
DNA. Primers (short DNA fragments) containing sequences complementary to the target
region along with a DNA polymerase, after which the method is named, are key
components to enable selective and repeated amplification. As PCR progresses, the
DNA generated is itself used as a template for replication, setting in motion a chain
reaction in which the DNA template is exponentially amplified. PCR can be extensively
.modified to perform a wide array ofgenetic manipulations
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq
polymerase (an enzyme originally isolated from the bacterium Thermus aquaticus). This
DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks,
the nucleotides, by using single-stranded DNA as a template and
DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA
synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating
.and cooling the PCR sample through a defined series of temperature steps
In the first step, the two strands of the DNA double helix are physically separated at a
high temperature in a process called DNA melting. In the second step, the temperature is
lowered and the two DNA strands become templates for DNA polymerase to selectively
amplify the target DNA. The selectivity of PCR results from the use of primers that
arecomplementary to the DNA region targeted for amplification under specific thermal
.cycling conditions

72) PRIME
PRIME (PRobe Incorporation Mediated by Enzymes) is a molecular biology research
tool developed by Alice Y. Ting and the Ting Lab at MIT for site-specific labeling of
proteins in living cells with chemical probes.[1][2] Probes often have useful biophysical
properties, such as fluorescence, and allow imaging of proteins.[1] Ultimately, PRIME
enables scientists to study functions of specific proteins of interest.

73) Primer dimer

A Primer dimer (PD) is a potential by-product in PCR, a common biotechnological
method. As its name implies, a PD consists of primer molecules that have attached
(hybridized) to each other because of strings of complementary bases in the primers.
As a result, the DNA polymerase amplifies the PD, leading to competition for PCR
reagents, thus potentially inhibiting amplification of the DNA sequence targeted for
PCR amplification. In quantitative PCR, PDs may interfere with accurate
quantification.

Promoter bashing (74

Promoter bashing is a technique used in molecular biology to identify how certain regions
of a DNA strand, commonlypromoters, affect the transcription of downstream genes.
Under normal circumstances, proteins bind to the promoter and activate or repress
transcription. In a promoter bashing assay, specific point mutations or deletions are made
in specific regions of the promoter and the transcription of the gene is then measured.
The contribution of a region of the promoter can be observed by the level of transcription.
If a mutation or deletion changes the level of transcription, then it is known that that
region of the promoter may be a binding site or other regulatory element. [1][2][3]

Promoter bashing is often done with deletions from either the 5' or 3' end of the DNA
strand; this assay is easier to perform based on repeated restriction digestion and gelpurifying fragments of specific sizes. It is often easiest to ligate the promoter into the
reporter, generate a large amount of the reporter construct using PCR or growth in
bacteria, and then perform serial restriction digests on this sample. The ability of
upstream promoters can be easily assayed by removing segments from the 5' end, and
the same for the 3' end of the strand for downstream promoters.[4]
As the promoter commonly contains binding sequences for proteins affecting
transcription, those proteins are also necessary when testing the effects of the promoter.
Proteins which associate with the promoter can be identified using anelectrophoretic
mobility shift assay (EMSA), and the effects of inclusion or exclusion of the proteins with
the mutagenized promoters can be assessed in the assay. This allows the use of
promoter bashing to not only discover the location on the DNA strand which affects
transcription, but also the proteins which affect that strand. The effects of protein
interactions with each other as well as the binding sites can also be assayed in this way;
candidate proteins must instead be identified by protein/protein interaction assays
instead of an EMSA

pUC19 (75
pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers.
[1]

The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and

the abbreviation for the University of California, where early work on the plasmid series
had been conducted.[2] It is a circular double stranded DNA and has 2686 base pairs.
[3]

pUC19 is one of the most widely used vector molecules as therecombinants, or the

cells into which foreign DNA has been introduced, can be easily distinguished from the
non-recombinants based on colour differences of colonies on growth media. pUC18 is
.similar to pUC19, but the MCS region is reversed

76) Recombinase Polymerase Amplification

Recombinase Polymerase Amplification (RPA) is a single tube, isothermal alternative to

the Polymerase Chain Reaction (PCR).[1] By adding a reverse transcriptase enzyme to an
RPA reaction it can detect RNA as well as DNA, without the need for a separate step to
produce cDNA,.[2][3][4] Because it is isothermal, RPA reactions need much simpler equipment
than PCR, which needs a thermal cycler. Operating best at temperatures of 37-42 C and still
working, albeit more slowly, at room temperature means RPA reactions can in theory be run
quickly simply by holding a tube. This makes RPA an excellent candidate for developing lowcost, rapid, point-of-care molecular tests. A recent international quality assessment of
molecular detection of Rift Valley fever virus performed as well as the best RT-PCR tests,
detecting less concentrated samples missed by some PCR tests and an RT_LAMP test.
[5]

RPA was developed and launched by TwistDx Ltd. (formerly known as ASM Scientific Ltd),

a biotechnology company based in Cambridge, UK.

77) reverse northern blot

The reverse northern blot is a variant of a northern blot in which the nucleic acid immobilized
on a membrane is a collection of isolated DNA fragments rather than RNA, and theprobe is
.RNA extracted from a tissue and radioactively labelled
DNA microarrays use methods akin to the reverse procedure, in that they involve the use of
isolated immobilized DNA fragments, and hybridization with a probe made from cellular RNA.
Thus the reverse northern blot, though originally uncommon, enabled northern analysis to
evolve into gene expression profiling, in which many (possibly all) of the genes in an organism
.may have their expression monitored

Reverse transfection (78

Reverse transfection is the invention and development of a microarray-driven gene
expression system by Junald Ziauddin and David M. Sabatini in 2001.[1] As DNA are
printed on a glass slide for the transfection process (the deliberate introduction of nucleic
acids into cells) to occur before the addition of adherent cells, the order of addition of
DNA and adherent cells is reverse that of conventional transfection. Hence, the word
.reverse is used

Ribosomal RNA (rRNA) intergenic spacer analysis (RISA) (79

Ribosomal RNA (rRNA) intergenic spacer analysis (RISA) is a method of microbial
community analysis that provides a means of comparing differing environments or treatment
impacts without the bias imposed by culture- dependent approaches. This type of analysis is
often referred to as community fingerprinting. RISA involves PCR amplification of a region of
the rRNA gene operon between the small (16S) and large (23S) subunits called the intergenic
.spacer region ISR

By using oligonucleotide primers targeted to conserved regions in the 16S and 23S genes,
RISA fragments can be generated from most of the dominant bacteria in an environmental
sample. While the majority of the rRNA operon serves a structural function, portions of the
16S-23S intergenic region can encode tRNAs depending on the bacterial species. However
the taxonomic value of the ISR lies in the significant heterogeneity in both length and
nucleotide sequence. In RISA, we attempt to exploit the length heterogeneity of the ISR,
which has been shown to range between 150 and 1500 bp with the majority of the ISR
.lengths being between 150 and 500 bp
The resulting PCR product will be a mixture of fragments contributed by several dominant
community members. This product is electrophoresed in a polyacrylamide gel, and the DNA is
visualized following staining. The result is a complex banding pattern that provides a
community-specific profile, with each DNA band corresponding to a bacterial population on
the original assemblage

Ribosome profiling (80

Ribosome profiling, or Ribo-Seq, is a technique developed by Nick Ingolia and Jonathan
Weissman that uses specialized messenger RNA (mRNA) sequencing to determine
which mRNAs are being actively translated.[1] It produces a global snapshot of all
the ribosomes active in a cell at a particular moment. Consequently, this enables
researchers to identify the location of translation start sites, their distribution, and the
speed of the translating ribosomes.[2] Ribosome profiling involves similar sequencing
library preparation and data analysis to RNA-Seq, but unlike RNA-Seq, which sequences
all of the mRNA of a given sequence present in a sample, ribosome profiling targets only
mRNA sequences protected by the ribosome during the process of decoding by
translation

run-off transcription (81

A run-off transcription assay is an assay in molecular biology which is conducted in vitro to
identify the position of the transcription start site (+1) of a specific promoter along with its
accuracy and rate of in vitro transcription.[1] [2][3] Run-off transcription can be used to
quantitatively measure the effect of changing promoter regions on in vitro transcription levels,
[1][2][4]

Because of its in vitro nature, however, this assay cannot accurately predict cell-specific

gene transcription rates, unlike in vivo assays such as nuclear run-on. [1][2]
To perform a run-off transcription assay, a gene of interest, including the promoter, is cloned
into a plasmid[4] The plasmid is digested at a known restriction enzyme cut site downstream
from the transcription start site such that the expected mRNA run-off product would be easily
separated by gel electrophoresis.[1][2][4] DNA needs to be highly purified prior to running this
assay.[1] [2] To initiate transcription, radiolabeled UTP, the other nucleotides, and RNA

polymerase are added to the linearized DNA.[1][2] Transcription continues until the RNA
polymerase reaches the end of the DNA where it simply runs off the DNA template, resulting
in an mRNA fragment of a defined length.[1][2] This fragment can then be separated by gel
electrophoresis, alongside size standards, and autoradiographed. [1][2][4] The corresponding
size of the band will represent the size of the mRNA from the restriction enzyme cut site to the
transcription start site (+1).[4] The intensity of the band will indicate the amount of product
.mRNA produced

Sanger sequencing (82

Sanger sequencing is a method of DNA sequencing based on the selective incorporation
of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA
replication.[1][2] Developed by Frederick Sanger and colleagues in 1977, it was the most
widely used sequencing method for approximately 25 years. More recently, Sanger
sequencing has been supplanted by "Next-Gen" sequencing methods, especially for
large-scale, automated genome analyses. However, the Sanger method remains in wide
use, primarily for smaller-scale projects and for obtaining especially long contiguous DNA
.sequence reads (>500 nucleotides)

Selection and amplification binding assay (83

Selection and amplification binding assay (SAAB) is a molecular biology technique
developed by T. Keith Blackwell and Harold M. Weintraub in 1990.[1] Its major use is to
.find the DNA binding site for proteins
Experimental details
SAAB experimental procedure consists of several steps, depending upon the knowledge
available about the binding site. A typical SAAB consists of the following steps
I. Synthesis of template with random sequence in binding site Three situations are
possible: (i) when both the binding site and the protein are known and available; (ii) when
only a consensus binding site is available and the binding protein is not; and (iii) when
the protein is available, but the binding site is unknown. When the binding site is not
known, the number of random nucleotide positions in the template must be large
II. Incubate labeled double stranded template with protein Usually the protein has to
synthesis in a host cell with fusion techniques. Longer incubation time and large quantity
are provided in case of unspecific binding site.[2]
III. Isolate the DNA bound protein by EMSA The DNA bound protein, migrated in
acrylamide gel is isolated by autoradiography as per Electrophoretic mobility shift assay
(EMSA) protocol.[3]

IV. Amplify the bound template by PCR. For positive control, amplify the starting template
.also The bound DNA is isolated via gel excision and purified, and amplified using PCR
V. Label amplified binding site and reselect for binding by EMSA (Usually 5 times)
Precede the binding step at least for 5 times with the amplified labeled DNA sample and
.fusion protein
VI. Sequence the DNA After final step of selection and electrophoresis, clone the DNA
into some cloning vector and sequence it. Originally Blackwell used Pyro sequencing,
which can be replaced by modern techniques

Sono-Seq (84
Sono-Seq (Sonication of Cross-linked Chromatin Sequencing) is a method in molecular
biology used for determining the sequences of those DNA regions in the genome near
regions of open chromatin of expressed genes. It is also known as "Input" in the ChipSeq protocol, since it follows the same steps except it doesn't requireimmunoprecipitation

Southern blot (85

A Southern blot is a method used in molecular biology for detection of a specific DNA
sequence in DNA samples. Southern blotting combines transfer of electrophoresisseparated DNA fragments to a filter membrane and subsequent fragment detection
.by probe hybridization
The method is named after its inventor, the British biologist Edwin Southern.
[1]

Other blotting methods (i.e., western blot,[2] northern blot, eastern blot, southwestern

blot) that employ similar principles, but using RNA or protein, have later been named in
reference to Edwin Southern's name. As the technique was eponymously named,
Southern blot is capitalized as is conventional for proper nouns. The names for other
.blotting methods may follow this convention, by analogy

Southwestern blotting (86

Southwestern blotting, based along the lines of Southern blotting (which was created
by Edwin Southern) and first described by B. Bowen, J. Steinberg and colleagues in
1980,[1] is a lab technique which involves identifying and characterizing DNA-binding
proteins (proteins that bind to DNA)[2] by their ability to bind to specific oligonucleotide

probes. The proteins are separated by gel electrophoresis and are subsequently
.transferred to nitrocellulose membranes similar to other types of blotting
The name southwestern blotting is based on the fact that this technique detects DNAbinding proteins, since DNA detection is by Southern blotting and protein detection is
.bywestern blotting
However, since the first southwestern blottings, many more have been proposed and
discovered. The former protocols were hampered by the need for large amounts
.of proteinsand their susceptibility to degradation while being isolated
Southwestern blot mapping" is performed for rapid characterization of both DNA-binding "
proteins and their specific sites on genomic DNA. Proteins are separated on
apolyacrylamide gel (PAGE) containing sodium dodecyl sulfate (SDS), renatured by
removing SDS in the presence of urea, and blotted onto nitrocellulose by diffusion. The
genomic DNA region of interest is digested by restriction enzymes selected to produce
fragments of appropriate but different sizes, which are subsequently end-labeled and
allowed to bind to the separated proteins. The specifically bound DNA is eluted from each
individual protein-DNA complex and analyzed by polyacrylamide gel electrophoresis.
Evidence that tissue-specific DNA binding proteins may be detected by this technique
has been presented. Moreover, their sequence-specific binding allows the purification of
the corresponding selectively bound DNA fragments and may improve protein-mediated
.cloning of DNA regulatory sequences

Stable-isotope probing (87

Stable-isotope probing is a technique in microbial ecology for tracing fluxes
of nutrients in biogeochemical cyling by microorganisms. A heavier stable isotope is can
be enriched in a substrate that is consumed by the organisms to be
studied. Biomarkers with the heavier isotope incorporated into them can be separated
from biomarkers containing the more naturally abundant lighter isotope by isopycnic
centrifugation. For example, 13CO2 can be used to find out which organisms are
actively photosynthesizing or consuming new photosynthate. As the
biomarker, DNA with 13C is then separated from DNA with 12C by
centrifugation. Sequencing the DNA identifies which organisms were consuming
existing carbohydrates and which were using carbohydrates more recently produced
.from photosynthesis

staggered extension process (88

The staggered extension process (also referred to as StEP) is a common technique used
in biotechnology and molecular biology to create new, mutated genes with qualities of
.one or more initial genes
The technique itself is a modified polymerase chain reaction with very short
(approximately 10 seconds) cycles. In these cycles the elongation of DNA is very quick
(only a few hundred base pairs) and synthesized fragments anneal with complementary
fragments of other strands. In this way, mutations of the initial genes are shuffled and in
the end genes with new combinations of mutations are amplified. [1][2]
The StEP protocol has been found to be useful as a method of directed evolution for the
discovery of enzymes useful to industry

Strep-tag (89
The Strep-tag system is a method which allows the purification and detection
of proteins by affinity chromatography. The Strep-tag is a synthetic peptide consisting of
eightamino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). This peptide sequence exhibits
intrinsic affinity towards Strep-Tactin, a specifically engineered streptavidin and can be Nor C- terminally fused to recombinant proteins. By exploiting the highly specific
interaction, Strep-tagged proteins can be isolated in one step from crude cell lysates.
Because the Strep-tag elutes under gentle, physiological conditions it is especially suited
for generation of functional proteins

Streptamer (90
The Streptamer technology allows the reversible isolation and staining of antigenspecific T-cells. This technology combines a current T-cell isolation method with
the Strep-tagtechnology. In principle, the T-cells are separated by establishing a specific
interaction between the T-cell of interest and a molecule that is conjugated to a marker,
which enables the isolation. The reversibility of this interaction and the fact that it can be
performed at low temperatures is the reason for the successful isolation and
characterization of functional T-cells. Because T-cells remain phenotypically and
functionally indistinguishable from untreated cells, this method may offer new strategies
in clinical and basic T-cell research

subcloning (91
In molecular biology, subcloning is a technique used to move a particular gene of interest
.from a parent vector to adestination vector in order to further study its functionality

.Subcloning is not to be confused with molecular cloning, a related technique

Surround optical fiber immunoassay (92

Surround optical fiber immunoassay (SOFIA) is an ultrasensitive, in vitro diagnostic
platform incorporating a surround optical fiberassembly that
captures fluorescence emissions from an entire sample. The technology's defining
characteristics are its extremely high limit of detection, sensitivity, and dynamic range.
SOFIAs sensitivity is measured at the attogram level (1018g), making it about one billion
times more sensitive than conventional diagnostic techniques. Based on its enhanced
dynamic range, SOFIA is able to discriminate levels of analyte in a sample over 10 orders
of magnitude, facilitating accurate titering.[1]
As a diagnostic platform, SOFIA has a broad range of applications. Several studies have
already demonstrated SOFIAs unprecedented ability to detect naturally
occurring prions in the blood and urine of disease carriers.[2][3][4] This is expected to lead to
the first reliable ante mortem screening test for vCJD, BSE, scrapie, CWD, and
other transmissible spongiform encephalopathies.[5]Given the technologys extreme
sensitivity, additional unique applications are anticipated, including in vitro tests for other
neurodegenerative diseases, such as Alzheimers and Parkinsons

Suspension Array Technology (93

Suspension Array Technology (or SAT) is a high throughput, large-scale, and multiplexed
screening platform used in molecular biology. SAT has been widely applied
togenomic and proteomic research, such as single nucleotide polymorphism (SNP)
genotyping, genetic disease screening, gene expression profiling, screening drug
discovery and clinical diagnosis.[1][2][3] SAT uses microsphere beads (5.6 um in diameter) to
prepare arrays. SAT allows for the simultaneous testing of multiple gene variants through
the use of these microsphere beads as each type of microsphere bead has a unique
identification based on variations in optical properties, most common is fluorescent
colour. As each colour and intensity of colour has a unique wavelength, beads can easily
be differentiated based on their wavelength intensity. Microspheres are readily
suspendable in solution and exhibit favorable kinetics during an assay. Similar to flat
microarrays (e.g. DNA microarray), an appropriate receptor molecule, such as
DNA oligonucleotide probes,antibodies, or other proteins, attach themselves to the
differently labeled microspheres. This produces thousands of microsphere array
elements. Probe-target hybridization is usually detected by optically labeled targets,
which determines the relative abundance of each target in the sampl

synchronous or synchronized culture (94

A synchronous or synchronized culture is a microbiological culture or a cell culture that
contains cells that are all in the same growth stage.
Since numerous factors influence the cell cycle, some of them stochastic (random), normal,
non-synchronous cultures have cells in all stages of the cell cycle. Obtaining a culture with a
unified cell-cycle stage is very useful for biological research. Since cells are too small for
certain research techniques, a synchronous culture can be treated as a single cell; the
number of cells in the culture can be easily estimated, and quantitative experimental results
can simply be divided in the number of cells to obtain values that apply to a single cell.
Synchronous cultures have been extensively used to address questions regarding cell cycle
and growth, and the effects of various factors on these.
Synchronous cultures can be obtained in several ways:

External conditions can be changed, so as to arrest growth of all cells in the culture,
and then changed again to resume growth. The newly growing cells are now all starting
to grow at the same stage, and they are synchronized. For example,
for photosynthetic cells light can be eliminated for several hours and then re-introduced.
Another method is to eliminate an essential nutrient from the growth medium and later to
re-introduce it.

Cell growth can also be arrested using chemical growth inhibitors. After growth has
completely stopped for all cells, the inhibitor can be easily removed from the culture and
the cells then begin to grow synchronously. Nocodazole, for example, is often used in
biological research for this purpose.

Cells in different growth stages have different physical properties. Cells in a culture
can thus be physically separated based on their density or size, for instance. This can be
achieved using centrifugation (for density) or filtration (for size).

In the Helmstetter-Cummings technique, a bacterial culture is filtered through a

membrane. Most bacteria pass through, but some remain bound to the membrane. Fresh
medium is then applied to the membrane and the bound bacteria start to grow. Newborn
bacteria that detach from the membrane are now all at the same stage of growth; they
are collected in a flask that now harbors a synchronous cultur

95) TA cloning
TA cloning is a subcloning technique that avoids the use of restriction enzymes[1] and
is easier and quicker than traditional subcloning. The technique relies on the ability
ofadenine (A) and thymine (T) (complementary basepairs) on different DNA
fragments to hybridize and, in the presence of ligase, become ligated
together. PCR products are usually amplified using Taq DNA polymerase which
preferentially adds an adenine to the 3' end of the product. Such PCR amplified
inserts are cloned into linearized vectors that have complementary
3' thymine overhangs.

toeprinting assay (96

The toeprinting assay (originally called extension inhibition assay(1)) is method to look at
the interaction of messenger RNA with ribosomes or other RNA-binding proteins. It is
different from standard DNA footprinting assay. The toeprinting assay identifies only one
edge of the complex on the RNA, whereas DNA footprinting identified both edges of the
complex on the DNA. The toeprinting assay has been utilized to examine the formation of
the translation initiation complex. The general idea is that you take an RNA of interest
and mix it with the 30s subunit of the ribosome, initiator tRNA, a synthetic
DNA primer that hybridizes downstream of the binding site, the four deoxynucleotide
triphosphates, andreverse transcriptase (RT). RT will normally synthesize cDNA by
extending the primer to the 5' end of the RNA. However, a bound ribosome will block RT
from continuing, resulting in a shortened cDNA that is called a toeprint when observed on
a sequencing gel. The precise nucleotide position of the toeprint on the RNA can be
determined by running a DNA sequencing reaction generated with the same primer in
adjacent lanes

Transmission electron microscopy DNA sequencing (97

Transmission electron microscopy DNA sequencing is a singlemolecule sequencing technology that uses transmission electron microscopy techniques.
The method was conceived and developed in the 1960s and 70s,[1] but lost favor when
the extent of damage to the sample was recognized. [2]
In order for DNA to be clearly visualized under an electron microscope, it must be labeled
with heavy atoms. In addition, specialized imaging techniques and aberration
corrected optics are beneficial for obtaining the resolution required to image the labeled
DNA molecule. In theory, transmission electron microscopy DNA sequencing could
provide extremely long read lengths, but the issue of electron beam damage may still
.remain and the technology has not yet been commercially developed

UniVec (98
UniVec is a database that can be used to remove the vector contamination from the DNA
sequences

VectorDB (99
VectorDB was a database of sequence information for common vectors used
in molecular biology

western blot (100

The western blot (sometimes called the protein immunoblot) is a widely used analytical
technique used to detect specific proteins in a sample of tissue homogenate or extract. It
uses gel electrophoresis to separate native proteins by 3-D structure or denatured
proteins by the length of the polypeptide. The proteins are then transferred to a
membrane (typically nitrocellulose or PVDF), where they are stained
with antibodies specific to the target protein.[1][2] The gel electrophoresis step is included in
.western blot analysis to resolve the issue of the cross-reactivity of antibodies
There are now many reagent companies that specialize in providing antibodies
(both monoclonal and polyclonal antibodies) against tens of thousands of different
proteins.[3] Commercial antibodies can be expensive, although the unbound antibody can
be reused between experiments. This method is used in the fields of molecular

biology, immunogenetics and other molecular biology disciplines. A number of search

engines, such as CiteAb, are available that can help researchers find suitable antibodies
.for use in western blotting
Other related techniques include dot blot
analysis, immunohistochemistry and immunocytochemistry where antibodies are used to
detect proteins in tissues and cells by immunostaining, and enzyme-linked
.immunosorbent assay (ELISA)
The method originated in the laboratory of Harry Towbin at the Friedrich Miescher
Institute.[1] The name western blot was given to the technique by W. Neal Burnette[4] and is
a play on the name Southern blot, a technique for DNA detection developed earlier
by Edwin Southern. Detection of RNA is termed northern blot and was developed by
George Stark at Stanford

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