18 Flow Cytometry and Introduction to Molecular Pathology

Flow Cytometry
History

1950 Wallace Coulter

o
Technique for analyzing individual cells
o
Automated cell counting
1970 Computer science, laser technology,
monoclonal Ab production, cytochemistry,
fluorochrome chemistry
Flow cytometry
o

Basic Principle

Suspension add Ab with dye chamber with laser

light scatters or is absorbed absorbed light is reemitted as fluorescence signals picked up by
photodiodes computer

French-American-British vs. World Health Organization

FAB
Morphology, cytochemical stains
ALL = 3; AML = 7

WHO
Morphology, cytochemical stains,
flow cytometry, karyotype, molecular data
* CD cluster of differentiation
Panel

Pan(el) B cell: CD 19, CD 20, CD 22

Pan(el) T cell: CD 2, CD 3, CD 4 and/or CD 7
CD 45: differentiates hematologic malignancies from
other neoplasms
CD 34 and HLA-DR Ags: markers of hematopoietic
stem cells; quality assurance in bone marrow
transplant

Lymphoid Malignancies

B-cell neoplasm
o
Precursor B malignancy is 75% -85% of ALL
Use CD 10 and 19
o

T-cell neoplasm:
o
15% -25% of ALL
Use CD 7
o
o
Older males with high count and
mediastinal mass
Myeloid Malignancies

AML
Monocytic

Megakaryocytic

Erythroleukemia
Dyes used in Flow Cytometry

FITC
Fluorescein isothiocyanate

Texas red

PE
Phycorerythrin

CD 13, 33, 117

CD 4, 11b, 11c, 14, 36, 64 or 68
CD 41 and 61
CD 235 (glycophorin)

Detection of Residual Disease

Source of relapse

Less sensitive than PCR

CD 10, TdT, CD 34 in CSF is indicative of T-cell residual

disease
Lymphocyte subset enumeration

Monitoring CD4 T-cells in HIV

Use CD 38 on CD 8 T-cells

More superior than RNA viral load

DNA Ploidy

Cells + Ab with fluorochrome to DNA fluorescent

emission of dye bound to DNA are measured
(>0r = 10,000 cells) analyzed and compared with
known cells without malignant DNA

E.g., breast CA with negative nodes

Clinical Applications

Immunophenotyping
o
Acute leukemias
o
Chronic lymphoproliferative disorders
o
Malignant lymphomas

Monitor progress of patients after chemo and bone

marrow transplantation

Detection of residual disease

Efficacy of CA treatment

Causes of failure
1. Multiple drug resistance = overexpression of
glycoproteins used in cellular transport
2. Biochemical detoxification
3. DNA replication and repair
4. Others

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Role of Flow Cytometry in the Work-up of MDR in

Cancer Treatment
Study of expression of cell surface and intracellular

markers of MDR

Intracellular accumulation and efflux of

chemotherapeutic drugs

In-vitro prediction of tumor sensitivity to radiation

and anti-neoplastic drugs through Bromodeoxyuridine assay of proliferative survival (vs.
clonogenic assay of Hamburger and Salmon 1970)
Reticulocyte Enumeration

Why measure?
1. Dysfunctional BM reticulocytopenia
2. Functional BM reticulocytosis
3. Monitoring of BM regenerative activity
after chemotherapy and BM
transplantation
Why use FC in reticulocytes?
Count is precise

Divides population into mature and immature

reticulocyte fraction
Calculates reticulocyte maturation index

Platelet Function Analysis

Platelet receptor quantitation

o
Diagnosis of congenital platelet disorder
o
Detection of surface markers

Platelet-associated IgG quantitation

o
Immune thrombocytopenia
o
Platelet cross-matching
Fibrinogen receptor occupancy studies for monitoring

efficacy of platelet-directed anticoagulation in

thrombosis
Cytoplasmic calcium measurement

Platelet microparticles for assessment of

hypercoagulable states
Functional analysis of lymphocytes

Transfusion medicine: anti-IgG Rh sensitization

Diseases of the immune system:

Tyrosine phosphorylation
o
o
Calcium influx
o
Oxidative metabolism
o
Neoantigen expression
o
Cellular proliferation
Organ Transplantation

Pretransplant X-match

HLA antibody screening

Post-transfusion Ab monitoring

Ex vivo T-cell graft depletion

Post-transplantation evaluation of immune recovery

Graft rejection

GVHD

Graft vs. leukemia effect

Microbiology

Identification of culturable and unculturable microbes

through IgG

Antimicrobial susceptibility

Drug cytotoxicity

Study of heterogenous microbial population after

antimicrobial therapy

Introduction to Molecular Pathology

DNA Extraction
Definition
DNA extraction is the

isolation and purification of

DNA (deoxyribonucleic acid)
Examples

DNA extraction is used to isolate:

o
Mitochondrial DNA
o
Genomic DNA

DNA can be extracted from

o
Cells or tissues
o
Environmental samples

Non-Examples

DNA extraction is not used to:

o
Isolate proteins or RNA
o
Give information about gene expression
Downstream Applications

After DNA is extracted, it is used as a template in

further molecular techniques such as:
o
PCR (polymerase chain reaction)
RFLP (restriction fragment length
o
polymorphism)
Southern blotting
o
DNA Analysis

Downstream techniques can:

Reveal how organisms are related
o
o
Identify cryptic species
o
Locate mutations in DNA
Qiagen DNA Extraction
1. Tissue Lysis. Detergents lyse membranes.
Proteinase K breaks down proteins.
2. Sample loading onto column. Nucleic acids bind to
membrane and other components flow through.
3. Column washing. Removes any contaminants.
4. Elution of DNA. DNA is release from column and
ready for use in downstream applications.
Principles of RNA Isolation
Purpose

Isolating RNA is a crucial first step in any analysis,

including common applications such as:
o RT-PCR
o Northern blotting
o cDNA library construction

Cells contain:
o Proteins
o DNA
o RNA

tRNA

rRNA (majority of RNA)

mRNA (~1-5%) necessary for

transcriptional studies
(i.e., gene expression)

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Ambion Site

This link gives a summary on their kits, reagents used,

and principles on isolation
http://www.ambion.com/techlib/tb/tb_158.html

Common reagents include:

Saturated phenol
o
o
Acid phenol: chloroform
o
Phenol:chloroform : IAA
o
Guanidinium solution
(Importance is to remove proteins, DNA, and/or other
impurities in the cell.)
Phenol:Chloroform

Organic solvents that can trap hydrophobic

substances/crap:
1. Membrane lipids
2. Polypeptide sequences
3. Polysaccharides

Cause hydrophilic (nucleic acids) substances to

interact with aqueous state

Gel Electrophoresis of DNA

What is Gel Electrophoresis?

Electro flow of electricity; phoresis from the Greek

to carry across

A gel is a colloid (a suspension of tiny particles in a

medium occurring in a solid form), like gelatin
Gel electrophoresis refers to the separation of

charged particles located in a gel when an electric

current is applied

Charged particles can include DNA, amino acids,

peptides, etc.
Why do gel electrophoresis?

When DNA is cut by restriction enzymes, the result is

a mix of pieces of DNA of different lengths

It is useful to be able to separate the pieces i.e., for

recovering particular pieces of DNA, for forensic work
or for sequencing
What is needed?

Agarose a polysaccharide made from seaweed.

Agarose is dissolved in buffer and heated, then cools
to a gelatinous solid with a network of crosslinked
molecules

Some gels are made with acrylamide if sharper bands

are required.

Buffer (in this case, TBE)

o
The buffer provides ions in solution to
ensure electrical conductivity

Not only is the agarose dissolved in buffer,

but the gel slab is submerged (submarine
gel) in buffer after hardening
Also needed are a power supply and a gel chamber
o
Gel chambers come in a variety of models,
from commercial through home-made, and
a variety of sizes
o

How does it work?

DNA is an organic acid, and is negatively charged

(remember, DNA for Negative)

When the DNA is exposed to an electrical field, the

particles migrate toward the positive electrode

Smaller pieces of DNA can travel further in a given

time than larger pieces
Steps in running a gel

DNA is prepared by digestion with restriction

enzymes

Agarose is made to an appropriate thickness (the

higher the % agarose, the slower the big fragments
run) and melted in the microwave

The gel chamber is set up, the comb is inserted

The agarose may have a DNA dye added (or it may

be stained later). The agarose is poured onto the gel

block and cooled

The comb is removed, leaving little wells and buffer

is poured over the gel to cover it completely.

The DNA samples are mixed with a dense loading dye

so they sink into their wells and can be seen
The DNA samples are put in the wells with a

micropipette
Micropipettes have disposable tips and can accurately

measure 1/1,000,000 of a litre

The power source is turned on and the gel is run. The

time of the run depends upon the amount of current
and % gel, and requires experimentation

At the end of the run the gel is removed (it is actually

quite stiff)

The gel is then visualized UV light causes the bands

of DNA to fluoresce

Many samples can be run on one gel but it is

important to keep track
Most gels have one lane as a DNA ladder DNA

fragments of known size are used for comparison

The DNA band of interest can be cut out of the gel ad

the DNA extracted

Or DNA can be removed from the gel by

Southern blotting
Polymerase Chain Reaction (PCR)
DNA

DNA is a nucleic acid that is composed of two

complementary nucleotide building block chains
The nucleotides are made up of a phosphate group, a
five-carbon sugar, and a nitrogen base
o DNA sugar deoxyribonucleic acid
o RNA sugar ribonucleic acid
DNA has four nitrogen bases
o Two are purines (2-ringed base)

Adenine (A), Guanine (G)

o Two are pyrimidines (1-ringed base)

Cytosine (C), Thymine (T)

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o
o

These four bases are linked in a repeated

pattern by hydrogen bonding between the
nitrogen bases.
The linking of the two complementary
strands is called hybridization.
A purine always links with a pyrimidine base
to maintain the structure of DNA.

Adenine (A) binds to Thymine (T),

with two hydrogen bonds
between them.

Guanine (G) binds to Cytosine (C),

with three hydrogen bonds
between them.
Example of bonding pattern:

Primary strand
C C GA A T GGGA T GC
GGC T T A C C C T A C G
Complementary strand

PCR

PCR is a technique that takes a specific sequence of

DNA of small amounts and amplifies it to be used for
further testing.
Targets. The targets in PCT are the sequences of DNA
on each end of the region of interest, which can be a
complete gene or small sequence.
The number of bases in the target can vary.
o
TTAAGGCTCGA...AATTGGTTAA
The represents the middle DNA
sequence, and does not have to be known
to replicate it.
Denaturing. Denaturing is the first step in PCR, in
which the DNA strands are separated by heating to
95C.
Primers. Primers range from 15 to 30 nucleotides, are
single-stranded, and are used for the complementary
building blocks of the target sequence.
o
A primer for each target sequence on the
end of your DNA is needed .This allows both
strands to be copied simultaneously in both
directions.
The primers are added in excess so they will
o
bind to the target DNA instead of the two
strands binding back to each other.
Annealing. Annealing is the process of allowing two
sequences of DNA to form hydrogen bonds.
o
The annealing of the target sequences and
primers is done by cooling the DNA to 55C.
o
PCR Taq DNA Polymerase

Taq stands for Thermus aquaticus,

which is a microbe found in 176F
hot springs in Yellow Stone
National Forest.

Taq produces an enzyme called

DNA polymerase that amplifies
the DNA from the primers by the
polymerase chain reaction, in the
presence of Mg.
Cycles.
o
Denaturalization: 94- 95C
o
Primer Annealing: 55- 65C
o
Extension of DNA: 72
o
Number of Cycles: 25-40
(See page 11.)

Requirements.
o
Magnesium chloride: .5-2.5mM
Buffer: pH 8.3-8.8
o
o
dNTPs: 20-200M
o
Primers: 0.1-0.5M
DNA Polymerase: 1-2.5 units
o
Target DNA: 1 g
o

Applications of PCR

Neisseria gonorrhea and Chlamydia trachomatis are

two of the most common sexually transmitted
diseases. The infections are asymptomatic and can
lead to pelvic inflammatory disease, salpingitis in
women, epididymitis in men, infertility, and ectopic
pregnancy.
o
Specimens include endocervical swabs,
urethral swabs, and urine samples.
o
The swabs are placed in a vial with
transport buffer containing 50mM MgCL2
and sodium azide as a preservative.
o
The swab specimens can be stored 2-30C
for 4 days or frozen at -20C.
The urine samples are refrigerated at 2-8C
o
or stored at -20C.
o
A target sequence is chosen for both,
amplified with polymerase, and then
evaluated with an enzyme immunoassay.

HIV-1 and Factor V Leiden also have a specific target

sequence amplified, and then quantitated by using a
microwell probe, horse-radish peroxidase enzyme,
and chromogen substrate.

The HIV-1 test is used as a monitor of the severity of

the virus. The HIV-1 causes a depletion of CD4+ T
lymphocytes, causing immunodeficiency, multiple
opportunistic infections, malignancies, and death.
o
The HIV-1 specimen is plasma collected in
EDTA that must be separated from the cells
within 6 hours.
o
Heparin cannot be used as an anticoagulant
because it inhibits PCR.
o
A 142 base target sequence in the HIV-1 gag
gene is converted from RNA to
complementary DNA, and to double
stranded DNA using Thermus thermophilus
DNA polymerase in the presence of
manganese and buffers, which performs the
reverse transcription and the amplification
steps simultaneously.
o
The standard specimen procedure can
quantitate HIV-1 RNA in a range of 40075,000 copies/mL.

Factor V Leiden is the Factor V in the coagulation

cascade.
o
Factor V is a genetic point mutation that
causes increased risk of life-threatening
blood clots.
o
The mutation causes the Factor V molecule
to be unresponsive to the natural anticoagulant protein C.
o
Factor V Leiden shifts the patients
hemostatic balance to thrombosis.
o
Factor V mutation gives an increased risk of
venous thrombosis in a homozygous

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person, during pregnancy, surgery, or while

using oral contraceptives.
Thrombosis - is the development of a blood
o
clot that occurs in 20-40% of patients with
venous thrombosis.
Thrombophilia - a tendency towards
o
clotting that occurs in 40-65% of adults with
unexplained thrombophilia.
o
Protein C - a naturally occurring
anticoagulant that occurs in 95-100% of
people with activated protein C resistance.
o
Treatment for patients with Factor V Leiden
mutations are to give lifelong coumadin.
Women with the mutation should not take
o
oral contraceptives, and they have
increased risk of thrombosis during
pregnancy.
PCR can also be used in forensic testing.
o
The DNA sequences used are of short
repeating patterns called VNTR (variable
number of tandem repeat), which can range
from 4 to 40 nucleotides in different
individuals.
o
One set of VNTR locus are inherited from
the mother and one set from the father.
o
The genes are amplified using PCR, and
then run through electrophoresis.
o
The position of the two bands on the
electrophoresis gel depends on the exact
number of repeats at the locus.

Three VNTR loci from suspects, along with

the DNA from the scene are run through
PCR amplification, and then through
electrophoresis.
This gives six bands, which can have
common bands for some individuals, but
the overall pattern is distinctive for each
person.

Extraction of DNA for Factor V

The anticoagulant tube with the patients blood

sample should be centrifuged to separate it into the
layers of plasma, Buffy coat, and the RBCs.

The buffy coat is used for the extraction because it

contains WBCs, which are nucleated and possess the
DNA.

Extract and discard plasma, taking care not to remove

the buffy coat.

Carefully extract 200l of buffy coat from each

sample and place in designated tube.

Add 25l of protease to each tube.

Add 200l of lysis buffer to each tube.

Vortex each tube for 15 sec. to ensure proper mixing.

Incubate each tube for 10 min. at 56C.

Centrifuge each to remove any mixture that may be

on the lid.

Add 210l of ethanol, vortex and then centrifuge

again.

Add sample mixture to column tube and centrifuge

for 1 min.

The column portion is inserted into a new tube and

washed twice, each time 500l of buffer is used to
elute substances adsorbed to the column that are not
DNA.

The column portion is then centrifuged for 1 minute

to remove excess washing buffer.

Next, 100L of eluting buffer is added.

This is incubated for 5 minutes at 25C, and then

centrifuged.

The elute is kept this time because it contains the

DNA.
During the 5 minute incubation, the master mix

should be prepared.
Master Mix
o
10x Buffer - 10 l
o
MgCl2 - 6 l
o
dNTP mix - 0.8 l of each nucleotide
o
F5F primer - 2 l
o
F5R primer - 2 l
Taq polymerase - 0.5 l
o
o
Sterile H2O - 73.7 l

Place 5l of patient sample and 95l of master mix in

vials and place these vials in a PCR panel, which will
then be placed in the thermocycler for the DNA
amplification cycles.

To prove that the DNA was amplified, a DNA enzyme

immunoassay (DEIA) is performed.

The test is done by denaturing the amplified DNA and

adding it to probe-coated microtiter wells.

If the amplified DNA sequences are complementary

to the probes, double stranded hybrids will form.

A mouse monoclonal antibody is added that will only

bind to double-stranded DNA hybrids.

Positive and negative wells are detected colorimetric

by adding an enzyme (conjugated protein A with
horseradish peroxidase), substrate, and chromogen.

This is is incubated at room temp. away from light for

30 mins. to develop the color.

The color is then stopped, by the addition of 200l of

an acidic stop solution.

The plate is then placed in the automated reader,

where each well is read spectrophotometrically.
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Each well is read at 450nm and then at 630nm.

The difference between the two absorbance:
A at 450nm
- A at 630nm
_______ Final A value
A positive hybridization result is indicated by an
absorbance value greater than the mean negative
control plus 0.150 absorbance units.
The machine then gives you a read out, from which
you calculate the patients results.

Conclusion

PCR is not only vital in the clinical laboratory by

amplifying small amounts of DNA for STD detection,
but it is also important for genetic predisposing for
defects such as Factor V Leiden.

The PCR technology can also be employed in law

enforcement, genetic testing of animal stocks and
vegetable hybrids, and drug screening along with
many more areas.

Basic Steps in a DNA Probe Hybridization

Uses of Nucleic Acid-Based Tests

1. Diagnosis of genetic diseases
2. Characterization of malignancies
3. Forensic studies
4. Paternity testing
5. Detection and identification of infectious agents
Molecular Methods
Nucleic acid hybridization methods

Amplification methods

Sequencing
Enzymatic digestion of nucleic acid

Nucleic Acid Hybridization

For detection and identification of pathogens. DNA specimen is
denatured; labeled single-stranded piece of DNA is added
unique to the pathogen (probe) which can be detected
enzymatically or radiographically.

Hybridization Formats

Solution format. Probe and target nucleotide strands

are put together in a solution.

Solid support format. Either probe or target nucleic

acids can form complex to a solid support and still be
capable of forming duplexes with complementary
strands.

Southern hybridization. Target nucleic acid bands are

transferred to a membrane and exposed to probe
nucleic acid.

Sandwich hybridization. Two probes are used. One

unlabeled probe is attached to the solid support and
via hybridization captures the target nucleic acid.
The labeled second probe specific to the target
sequence detects the presence of the duplex.

In situ hybridization. Makes use of patients cells or

tissues as the solid support to detect a particular
pathogen. Nucleic acid of the pathogen is release and
denatured to a single strand with the base sequence
intact.
Principles of nucleic acid hybridization. Identification of
unknown organism is established by positive hybridization.
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DNA hybridization
of human
papilloma virus
probes.

Principle of the solution hybridization format.

Principle of hybridization
detection. Radioactive reporter
with hybridization detected by
autoradiography.

Probes labeled with biotin-avidin

reporter with hybridization
detected by a colorimetric assay.

Probes labeled with

chemiluminescent
reporter (acridinium) with
hybridization detected by
a luminometer to detect
emitted light.

Southern Blot. The DNA fragments from restriction

endonuclease treatment is transferred or blotted onto a nylon
membrane. The membrane is hybridized with specific labeled
probe containing the DNA sequence homologous to the one
sought.
Radiolabeled probe
detection of Herpes
virus. Autoradiogram
from specimens
containing Herpes
simplex 1 & 2
hybridized with 32Plabeled specific HSV1/HSV-2 probes.

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PCR Amplification

Types of Hybridization Assays

Amplification Methods

Target nucleic acid amplification PCR

o The most widely used target nucleic acid
amplification method
o Principle of complementary nucleic acid
hybridization with nucleic acid replication
applied repeatedly through numerous
cycles (30-50)

Three sequential reactions of PCR

1. Denaturation of target nucleic acid
2. Primer annealing to single-stranded target
nucleic acid
3. Extension of primer target duplex
RT-PCR Amplification

Polymerase Chain Reaction (PCR) basic principle.

Two oligonucleotide primers, 20 to 30 bases in length,
complementary to each one of the two strands of a DNA target
molecule, separated by approximately 200 to 400 bases. DNA is
first denatured by heat, the oligonucleotides and DNA (Taq)
polymerase are added to hybridize to C strands and elongate
respectively. Cycle repeated 30 to 40 times so that the target
DNA can be detected.

Detection of PCR Products

Amplicon PCR amplification product

Methods of detecting PCR products:

Solution or solid-phase formats with reporter
molecules that generate:
o Radioactive signal
o Colorimetric signal
o Fluorometric signal
o Chemiluminescent signal
Modifications of PCR Method

Multiplex PCR. More than one primer pair in PCT

mixture; internal controls
nd

Nested PCR. Sequential use of 2 primer sets; 2 set

of primers is internal to the first amplicon.

Quantitative PCR. Quantitate actual number of

targets originally in the specimen: infectious burden
(i.e., HIV)

RT-PCR. Amplifies an RNA target with the use of

reverse transcriptase.

Real-time PCR. Combines rapid thermocycling with

rapid detection of amplicons by fluorescent labeled
probes as the hybrids are formed in real time.

Arbitrary primed PCR. Uses short primers that are

not specifically C to a particular sequence
of a target DNA.
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PCR reaction products on agarose gel. The amplified DNA

strands in the PCR reaction mixture are separated
electrophoretically and stained with ethidium bromide for
visualization.
Automated PCR instrument. Includes a thermocycler and an
EIA reader to perform and read the result of a PCR that uses an
enzyme-labeled detection system.

Restriction fragment length polymorphism (RFLP) analysis.

Procedural steps for pulsed-field gel electrophoresis (PFGE)

Principle of DNA enzymatic digestion and gel electrophoresis.

Nucleic Acid Probe Amplification

Amplification of probe nucleic acid rather than the

target nucleic acid
Ligase Chain Reaction (LCR) amplified probe is the

final reaction product to be detected

Amplification of probe signal signal used to detect

hybridization between amplicon and probe can be
amplified by increasing the number of reporter
molecules per probe.

DNA restriction enzymes and their specificities.

Enzymes are named for the bacteria from which they are
isolated (N is any base and N, its pairing counterpart.)

Ligase chain reaction (LCR) or oligonucleotide ligation

amplification (OLA). Two oligonucleotides homologous to
adjacent sequences on the target DNA are joined together by
the ligase only when their ends are brought into close proximity
by hybridization of the template DNA. Once the ligase has
connected the two oligonucleotides, the product of ligation is
denatured from the target DNA so that both the target DNA
and the ligated primers can serve again as targets for
amplification.

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Q Replicase. Q replicase amplifies the signal of the probe

and not the target nucleic acid itsef. The probe is an RNA
molecule, midivariant (MDV-1) and a specific RNA fragment
corresponding to a complementary piece of RNA or DNA in the
organism to be detected. The probe RNA binds to the
complementary target, and after removing the unbound MDV-1
molecules, the Q replicase is added to replicate the RNA
probe.

Q amplification for HIV-1 infected cells detected by

fluorescent label. Very low numbers of HIV-1 infected cells can
be detected.
Nucleic Acid Sequencing

Determine the exact nucleotide sequence of a gene

or gene fragment obtained from an organism.

Uses:
1. Detecting and classifying previously
unknown human pathogens
2. Identifying various known microbial
pathogens and their subtypes
3. Determine which specific nucleotide
changes resulting from mutations are
responsible for antibiotic resistance
4. Establishing relatedness between isolates of
the same species
Enzymatic Digestion and Electrophoresis of Nucleic Acids

Uses endonucleases recognize a specific nucleotide

sequence (usually 4-8 nucleotides in length)

The longer the strand, the greater the likelihood of

more recognition sites and thus more fragments.

Plasmid Fingerprinting

Herpes simplex virus DNA

restriction patterns. DNA
from different herpes simplex
virus isolates was cleaved
with 3 different restriction
endonucleases,
electrophoresed on an
agarose gel, and stained with
ethidium bromide.

DNA sequencing apparatus. Four DNA oligonucleotides

complementary to one of the strands are synthesized using
specific DNA primers, ddNTP (dideoxynucleoside triphosphate),
and DNA polymerase. The DNA sequence of 300 and 500 base
fragment can be determined.

Labeled DNA fragments from a sequencing

reaction. For identification of specific
microbial isolates and for the
determination of the genetic relatedness
between different microorganisms.

High-Density DNA Probe

High-density oligonucleotide probe array

Hybridization of fluorescent-labeled nucleic acid

target to large sets of oligonucleotides synthesized at
precise locations on a miniature glass substrate or
chip

Uses:
o
Pathogen identification and classification
o
Polymorphism detection
o Drug resistance mutations for viruses (HIV)

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