Molecular Biotechnology

C G V ipin Shankar
Fundamentals of Molecular
Biotechnology

 Revolutionary scientific discipline based on the ability to

transfer specific units of genetic information from one
organism to another.

 Relies on the techniques of genetic engineering -

Recombinant DNA Technology (RDT)
Aims of Molecular
Biotechnology

 To create a useful product or a commercial

process, by choreographing the various Molecular
Waltzes of life, taking place largely inside the cells.
 Biotechnology is a term, that covers various
techniques for using the properties of living
things to provide products & services.
 Biotechnology operates at the
molecular levels of life, where the
seemingly solid boundaries between
species disappear.

Down among those molecules there

lies little difference between a man
and a bacterium.

What biotechnology does is to

choreograph the complex dances
among the molecules that ultimately
make every living thing what it is.
The Molecular Biotechnology
Revolution

 Molecular biotechnology is only the current

chapter in a story that began long time ago.
 In the modern context neither bread making or
wine fermentation, nor genetic alteration by
selective breeding, or plant cloning by grafting,
or the use of microbial products in fermentation;
are considered as Biotechnology
The Molecular Biotechnology
Revolution…

 What is new about modern biotechnology is not

the principle of using various organisms but the
techniques for doing so.
 Biotechnology has produced so many
applications very rapidly.
In the beginning

 1665 : Robert Hooke

and the Onion peel –
‘The Cells’.
 1675 : Anton Van
Leeuwenhoek and the
microscopes – shape &
size of various
microscopic particles.
As the years passed….
 1839 : Matthias Schleiden
& Theodore Schwann –
‘The Cell Theory’.
 1859 : Darwin the
Voyager – ‘On the origin
of species by natural
selection’.
 1866 : Mendel the Monk
- ‘Genetics and
Mendelism’.
As the years passed…

 1869 : Johann Miescher – Discovery of DNA

 1900 : Rediscovery of Mendelism
 1902 : Walter Sutton – Genes present on
Chromosomes.
 1910 : Morgan and the fruit flies.
 1928 : Griffith – ‘The transforming element’.
As the years passed…
As the years passed…
 1941 : Beedle & Tatum-
‘one gene one enzyme’.
 1944 : Oswald Avery –
DNA the genetic carrier.
 1953 : Watson and Crick –
Structure of DNA
As the years passed…
 1967 : Har Gobind Khorana & Marshal
Nirenberg – The Genetic Code.
 Molecular Biotechnology Now

Micro- Bio- Immunol Chemical

Genetics Engineeri
biology chemistry ogy
ng
Molecular

Biology
Biology

Cell
Molecular Biotechnology

Crops Drugs Vaccines Diagnostics Livestock

Basic concept of RDT

 Techniques for
 Identifying (locating),

 Isolating,

 Altering &

 Studying

DNA segments
D (daring) N (nucleotide) A (adventures)
Techniques for handling DNA

 Preparation of pure samples of DNA.

 Cutting DNA molecules.
 Analysis of DNA fragment sizes.
 Joining DNA molecules together.
 Introduction of DNA into host cells.
 Identification of cells that contain recombinant
DNA molecule.
Vehicles for carrying DNA

 Plasmids.
 Phages.
 Viruses.
Plasmids

 Circular DNA molecules of independent

existence in the bacterial cell.
 Carry one or more gene.
 Carry selectable markers.
 Have at least one origin of replication
 May exist as episomes
 Vary in size and copy number.
 Various incompatibility groups exist.
Plasmids…

 Classified into
 Fertility of F Plasmids.

 Resistance or R Plasmids.

 Col Plasmids.

 Degradative Plasmids.

 Virulence Plasmids.
Bacteriophages
 DNA hijackers.  Have protective capsids.
 Viruses infecting bacteria.  Lytic & Lysogenic life cycles.
Viruses

 Absence of plasmids in higher cells limit use of

plasmids and phages as vectors.
 Have considerable potential as cloning vehicles in
animal cells.
Purification of DNA from living
cells

 Three distinct kinds of DNA usually isolated

 Total cell DNA.

 Pure Plasmid DNA.

 Phage DNA.
Preparation of Total Cell DNA

 The cells are grown and harvested.

 The cells are broken open to release their
contents.
 The cell extract is treated to remove all
components except the DNA.
 The resulting DNA solution in concentrated.
Preparation of cell extract
 Physical methods
 Mechanical force
 Grinding
 Centrifugation
 Enzymatic method
 Lysozyme
 Lysozyme + EDTA

 Chemical methods
 Ethylene di-amine tetra
acetate (EDTA).
 Detergents – SDS
 Cetyl tri-methyl
ammonium bromide
(CTAB).
Purification of DNA from crude
extract

 Deproteinization
 1:1 mixture of phenol and chloroform

 Protease treatment (Pronase, Proteinase K)

 Ribonuclease treatment to degrade RNA

 Anion exchange resins.
Concentration of DNA

 Ethanol precipitation.
 In the presence of monovalent cations like Na+, at
temperature – 20 oC or less.
 Sticks to glass rod.
 Leaves short chain and monomeric nucleic acids
in solution.
 Added advantage of removing RNA traces.
Measurement of DNA
concentration

 Ultra violet absorbance spectrophotometry.

 Measured at 260 nm.

260 of 1.0 corresponds to 50 µg of ds DNA.

 A

 Purity of DNA can also be assayed.

 Pure sample has (A
260 / A280) = 1.8.

 Ratios of less than 1.8 indicate contaminations by

proteins or phenol.
Preparation of Plasmid DNA

 Similar to Genomic DNA isolation.

 Physical differences between genomic and
plasmid DNA exploited for separation.
 Differences in size and conformation.
 Plasmids are very small compared to genomic DNA.
 After extraction plasmids remain circular while
genomic DNA is broken and occur in linear
conformation.
Separation on the basis of
size

 Cells lysed under specific conditions –

 EDTA + Lysozyme treatment in presence of sucrose.

 Sucrose prevents cell burst.

 Spheroblasts – partially wall-less cells that retain

genomic DNA, are formed.
 Cell lysis induced by non-ionic detergents (Triton X-
100).
 Debris removed by centrifugation.

 Clear lysate consisting entirely of plasmid DNA left.

Separation on the basis of
conformation

 Three different conformations

 Supercoiled DNA.

 Covalently closed-circular DNA.

 Open circular DNA.

 Plasmids exist as supercoils.

 Can be separated by –
 Alkaline denaturation.

 EtBr- CsCl density gradient centrifuagation.

Alkaline denaturation

 At a pH range of 12.0 – 12.5, non-supercoiled DNA is

denatured while supercoiled DNA is not.
 Cell extract (Preferably clear lysate) is adjusted to this pH
using sodium hydroxide.
 Causes unwinding of double helix and denaturation of
non-supercoiled DNA.
 Addition of acid to the mixture causes denatured DNA to
aggregate to form tangled masses- removed by
centrifugation.
 Supercoiled Plasmid DNA remains in solution.
EtBr - CsCl density gradient
centrifugation

 CsCl density gradient is prepared.

 DNA has a buoyant density of 1.7 g/cm3.
 EtBr binds to DNA by intercalating between
adjacent base pairs.
 Results in decrease in buoyant density.
 Linear DNA – decrease of 0.125 g/cm3.
 Supercoiled DNA – decrease of 0.085 g/cm3.
 Supercoiled DNA forms a different layer during
centrifugation
Preparation of phage DNA

 Extracellular medium of infected bacterium used

instead of cell extract.
 The phage culture is induced to undergo a lytic
phase to get a high titre phage concentration in
the extra cellular medium.
Visualizing DNA molecules in
a gel

 DNA carry a net negative charge – move

towards the positive pole.
 Can be separated on the basis of size on agarose
gel.
 Visualized by staining or autoradiography.
 Distance moved by molecule D = a-b (logM)
 Where a and b are constants depending on
electrophoretic conditions,
 M is the molecular mass
Manipulation of purified DNA

 Manipulation includes
 Cutting and joining DNA molecules,
 Shortening and lengthening of DNA molecules,
 Addition or removal of specific chemical groups.
 Provides foundation for
 Gene cloning,
 DNA biochemistry
 Study of gene structure
 Study of gene expression
 Carried out using specific enzymes.
DNA manipulative enzymes

 Nucleases
 Ligases
 Polymerases
 Modifying enzymes
 Topoisomerases
Nucleases

 Degrade nucleic acid molecules by breaking the

phosphodiester bonds .
 Two types
 Exonucleases – removes nucleotides one at a time
from the end of the nucleic acid molecule.
 Endonucleases – break internal phosphodiester bonds
within a nucleic acid molecule.
 Restriction endonucleases cleave at specific sites
 Three types – type I, type II and type III
Restriction digestion

 Type II enzymes used.

 Recognize specific recognition sequences and
cuts DNA at these sequences.
 Some produce blunt ends, while others yield
sticky ends.
 Have specific requirements of temperature and
Mg2+ concentration.
 Results in Star Activity if requirements are not
met properly.
Restriction mapping

 Relative positions of recognition sites of a number of

restriction enzymes on a DNA molecule.
 A series of restriction digestion performed on DNA and
fragments produced by each enzyme are determined by
electrophoresis.
 The fragments are then compared with size markers.
 This information is supplemented by a series of double
digestions.
Ligases

 Two phosphodiester bonds, one for each strand

is made to link two DNA molecules.
 Both blunt and sticky ends can be ligated.
 Sticky ends are ligated more efficiently.
 Adaptors, Linkers and homopolymer tailing are
used to generate sticky ends.
Polymerases

 Synthesize a new strand of DNA complimentary

to an existing DNA or RNA template.
 Requires a primer.
 DNA pol I has duel activity
 DNA polymerization
 DNA degradation (Nick translation)
 Klenow fragment – fragment of pol I retains the
polymerase activity but lacks nuclease activity.
DNA modifying enzymes

 Alkaline phosphatase – from E. coli or calf

intestinal tissue – removes phosphate group from
5’ terminus.
 Poly nucleotide kinase – from E. coli infected
with T4 phage – adds phosphate groups to free
5’ terminus.
 Terminal deoxy-nucleotidyl transferase – from
calf thymus tissue – adds one or more deoxy-
nucleotides on to the 3’ terminus.
Topoisomerases

 Change the conformation of covalently close-

circular DNA by introducing or removing
supercoils.
 Very important in replication.
 Use in genetic engineering debated.
Transformation – the uptake
of DNA by bacterial cells

 Uptake and stable retention of plasmid detected

by expression of genes carried by plasmid.
 Most species uptake DNA from media naturally
– particularly if the DNA molecule is a plasmid
with an origin of replication recognized by the
host.
 Competent cells have higher transformation
efficiency.
Preparation of competent E.
coli cells

 Cells soaked in ice cold 50 mM CaCl2 solution.

 CaCl2 precipitates DNA on cell surface.
 Actual uptake facilitated by heat shock at 42 0C.
 Heat shock produces transient pores on the cell
membrane.
Other methods for
transformation

 Transfection – recombinant phage DNA

packaged into phage heads and allowed to infect
cells.
 Protoplasts and Electroporation.
 Microinjection.
 Gene gun.
 Viral infection.
The problem of selection

 Ligation mixture contains

 Desired recombinant molecule.

 Unligated vector molecule.

 Unligated DNA fragments.

 Vector molecules that have re-circularized, without

new DNA being inserted (self ligated vector).
 Recombinant DNA molecules that carry the wrong
inserted DNA fragment.
 Success or failure of gene cloning
studies depends on whether or not a
strategy can be devised by which
clones of the desired gene can be
selected directly, or alternatively,
distinguished from other
recombinants.

Once this problem is resolved, and a

clone has been obtained, a wide
variety of information can be
extracted from the gene
Strategies for obtaining the
correct clone.

 Direct selection for the desired gene.

 Identification of clone from a gene library.
Direct Selection

 Selectable markers carried by recombinant

DNA.
 Antibiotic resistance, specific enzyme activity etc
used as markers.
 Insertional inactivation of selectable markers is
another approach.
 Insertional inactivation detected by replica
plating.
5-bromo, 4-chloro 3-indodyl – β D galactopyranoside
+ isopropyl thiogalactoside
Direct selection by marker
rescue

 Genes that code for biosynthetic enzymes are

used.
 A mutant host strain which lacks the specific
enzymatic activity is used (auxotrophic).
 The correct recombinant construct contains the
gene coding for the enzyme.
 Only those hosts that contain the correct DNA
construct (non – auxotrophic) can survive on a
minimal media.
Identification of gene from a
gene library

 A genomic library is a collection of clone

sufficient in number to be likely to contain every
single gene present in a particular organism.
 Total cell DNA is purified and partial restriction
digestion is carried out.
 Resulting fragments are cloned into suitable
vectors.
 cDNA libraries are much easier to handle.
c DNA libraries

 Not all genes are expressed at a particular time.

 Total mRNA copy of a cell at a time represents
the genes expressed.
 mRNA can be reverse transcribed to give cDNA.
 The resulting cDNA clones will represent the
mRNA present in the original preparation.
 A library of cDNA will be much smaller than a
genomic library
 Any two single-stranded nucleic acid
molecules have the potential to form base
pairs with one another.

With most pairs of molecules the resulting

hybrid structures are unstable, as only a
small number of individual inter-strand bonds
are formed.

However, if poly-nucleotides are

complementary then extensive base-pairing
will occur, to form a stable double stranded
molecule.

This can occur between ss DNA , ss RNA &

also between a ss DNA and an ss RNA
Colony & Plaque hybridization

 Colony or plaque transferred to nitro-cellulose

membrane.
 Membrane treated to remove all contaminating materials.
 DNA alone remains on membrane.
 DNA denatured to produce ss DNA molecules.
 ss DNA bound to nitro-cellulose membrane by raising
temperature to 80 0C.
 DNA binds to membrane through the sugar-phosphate
backbone, bases remain free to base pair.
Colony & Plaque
hybridization…

 Labeled probes added to membrane.

 Probes base pair with complementary regions.
 Labels can be radioactive, fluorescent (biotin,
avidin,) or chemi-luminescent (HRP, luminol).
Applications

 Abundancy probing to analyze a cDNA library.

 Oligonucleotide probes for genes whose
translation products have been characterized.
 Heterologous probing to identify related genes.
Locating the position of a
cloned gene on a DNA
molecule

 Restriction digest probed with labeled probe.

 Can be done when restriction fragments are still
on the gel.
 This is cumbersome and yields false positive
results.
 Hence restriction digest is transferred on to a
nitro-cellulose membrane – Southern transfer
Southern Blotting

 Perfected by Prof. E M Southern.

 DNA bands from gel transferred on to
membrane, using buffers.
 Same method can be used to transfer RNA
(Northern) and Protein (Western) molecules.
 Advanced methods couple electrophoresis with
blotting – electro blotting.
Separating large DNA
molecules by electrophoresis

 Orthogonal Field Alteration Gel Electrophoresis

(OFAGE).
 Contour Clamped Homogeneous Electric Field
(CHEF).
 Field Inversion Gel Electrophoresis (FIGE).
in situ Hybridization

 Provides a direct visual of a cloned gene on the

light microscopic image of a chromosome.
 Cells treated with a fixative to a glass slide.
 Treated with ribonuclease and NaOH to degrade
RNA and denature DNA.
 Base pairing between individual polynucleotide
strands is broken and chromosomes unpack to a
certain extent.
in situ Hybridization…

 A sample of cloned gene is then labeled and

applied to the chromosome preparation.
 Hybridization occurs and a dark spot results after
autoradiography.
 If a fluorescent marker is used the technique is
called FISH (Fluorescent in situ hybridization)
Chromosome walking

 When the position of the gene is known, but no

probe is available.
 A probe for a near by gene is used.
 Two clone libraries prepared with different
restriction enzymes required.
 The clone containing the known gene from
library A is used to hybridize library B.
 One of the positive hits in library B is used to
hybridize library A.
Chromosome walking…

 This process is continued, gradually building up

a partial restriction map of overlapping
fragments.
 Tedious technique.
 Longest walk ever carried out till date is only 250
kb in length.
Restriction Fragment Length
Polymorphism (RFLP)
 The coexistence of multiple alleles at a locus is
called genetic polymorphism. Any site at which
multiple alleles exist as stable components of the
population is by definition polymorphic.
 A change in a single nucleotide when alleles are
compared is called a single nucleotide
polymorphism (SNP).
RFLP…

 A difference in restriction maps between two

individuals is called RFLP.
 Basically a RFLP is a SNP that is located in the
target site for a restriction enzyme.
 It can be used as a genetic marker in exactly the
same way as any other marker.
Genetic fingerprinting

 Specialized form of RFLP.

 The mini satellite DNA is probed.
 This differs in length in different individuals.
 Length of repeats ranges from 9 to 40 bp.
 Number of mini satellite repeats ranges from 10
to 30.
 Variability due to loss or gain during replication.
Genetic fingerprinting…

 A mini satellite pattern (fingerprint) represents

the repertoire of the lengths of some of these
sequences in an individual.
 The chance of finding two individuals with the
same pattern is about one in 105 – 108.
Electro Motility Shift Assay
(EMSA)

 Used to identify DNA-protein interactions.

 Protein bound DNA has relatively lesser mobility
compared to free DNA.
 Can be used to determine DNA region binding
to protein.
 Identifies regulatory elements in genes.
DNA foot printing

 Interaction of regulatory protein protects DNA

from degradative action of endonucleases.
 The DNA fragment is end labeled with a
radioactive marker.
 The DNA is mixed with the protein.
 The mixture is then treated with DNase I.
 The length of DNA where the protein binds is
protected.
Hybrid release translation (HRT)
& Hybrid arrest translation
(HART)

 Used to identify translation product encoded by a cloned

gene.
 cDNA clone is prepares from mRNA.
 For HRT cDNA is immobilized on nitrocellulose
membrane and incubated with mRNA.
 Specific cDNA gets hybridized and remains bound to
membrane.
 These are now retrieved and translated in cell free system.
 The protein mixture is run on PAGE
HART…

 For HART, the cDNA is slightly denatured and

added to mRNA sample.
 Hybridization occurs, and this hinders translation
in a cell free system.
 Only non-hybridized cDNA undergoes
translation.
 So all proteins except that coded by the mRNA
is produced.
Polymerase Chain Reaction
(PCR)

 Selective amplification of chosen region of a

DNA molecule.
 The border sequences of the DNA of interest
should be known.
 Oligonucleotides complementary to the border
sequences act as primers.
 Taq polymerase is used.
Primer designing

 Complementary to the sequence flanking the

gene.
 The 3’ ends of the hybridized primers should
point towards one another.
 The gene should not be greater than 3kb and
ideally be less than 1kb.
 Length of primer should be considered.
 GC content should be high.
Correct temperatures to be
used
 Denaturation temperature – ~94 0C.
 Annealing / Renaturation temperature – ~ 45 –
55 0C.
 Extension / Synthesis temperature – ~75 0C.
 Annealing temperature should be less than Tm.
Tm = (4 x [G + C]) + (2 x [A + T]) 0C
Types of PCR

 Gene Synthesis PCR

 Overlapping primers used to produce desired DNA.

 Reverse Transcriptase PCR (RT – PCR)

 A reverse transcriptase step is included in the first step
followed by normal PCR.
 Yields cDNA from mRNA.
 Real Time PCR (RTPCR)
 Tagged primers used to determine concentration on
PCR product during reaction.
Random amplification of
polymorphic DNA (RAPD)

 Another method of genetic finger printing.

 Random primers used to hybridize with total
genomic DNA.
 PCR performed to amplify polymorphic DNA.
 Each primer produces unique pattern for each
individual organism.
 Usually used to differentiate between different
variety of plant cultivars.