Unit 2 Basic Immunologic
Reactions
Part 7 Molecular Techniques
Terry Kotrla, MS, MT(ASCP)BB
Molecular Diagnostic Assays
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Very powerful tools.
Quick supplies information to assist in
diagnosis and monitoring of diseases.
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Bacteria
Viruses
Genetic diseases
This technology is impacting every area of the
clinical laboratory.
Molecular Techniques
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Techniques include
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Enzymatic cleavage of nucleic acids
Gel electrophoresis
Enzymatic amplification of target sequences
Hybridization with nucleic acid probes
Advantages and disadvantages of each will be
discussed.
Gene
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Genes are located on 23
pairs of chromosomes.
DNA is packed into genes.
Genes are units of heredity
DNA organized into long
structures called
chromosomes, genes are
pieces of DNA which
correspond to a single unit
of inheritance.
Two types of nucleic acids: RNA &
DNA
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DNA carries genetic information within
chromosomes of each cell.
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Main role is long-term storage of information.
Blueprint to construct other cell components
RNA
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Transcribed from DNA
Central role is protein synthesis
DNA and RNA
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DNA is encoded with four interchangeable
"building blocks", called "bases",
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Adenine and Thymine, which pair together.
Cytosine and Guanine, which pair together.
RNA has five different bases:
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Adenine and Uracil, which pair together.
Cytosine and Guanine, which pair together.
Three Main Differences
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DNA is double stranded, RNA is single
stranded.
DNA contains deoxyribose, RNA contains
ribose.
Complementary base to adenine is thymine in
DNA and uracil in RNA.
RNA versus DNA Structure
DNA Replication
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DNA very stable.
Loses conformational structure under
extremities of heat, pH or presence of
destablizing agents.
Semi-conservative process, one strand acts as
template to create exact copy.
Bonds holding strands together are weak.
DNA Replication – In-Vivo
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Strand “unzips”, hydrogen bonds between base pairs are
broken.
Sequence of bases on strand serve as template to which
complementary bases are added.
When process is complete 2 identical DNA molecules are
formed.
DNA Replication – In-Vitro
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Two steps
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Denaturation – can use heat or alkaline solutions
to break bonds and separate the two strands, if
heat is used term is called “melting”.
Annealing – strands cool and complementary
strands spontaneously rejoin.
This process is exploited in the laboratory.
Types of RNA
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One strand of DNA serves as template for messenger
or “mRNA”.
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mRNA carries information from DNA to ribosome.
Uracil transcribed where Thymine would have occurred.
Transfer RNA (tRNA) transports amino acids to
make proteins.
Ribosomal RNA (rRNA) acts as site of protein
synthesis directed by mRNA.
RNA less stable and degrades more rapidly than
DNA.
Hybridization Techniques
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Spontaneous pairing of DNA strands forms
the basis for detection and characterization of
genes.
Probe technology used to identify individual
genes or DNA sequences.
Nucleic Acid Probes
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Nucleic acid probe is a short strand of DNA
or RNA of known sequence
Used to identify presence of complementary
single strand of DNA in patient sample.
Binding of the patient strand with the probe is
known as hybridzation.
Nucleic Acid Probes
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Two DNA strands must share at least 16 to 20
consecutive bases of perfect complementarity to
form stable hybrid.
Match occurring as a result of chance less than 1 in a
billion.
Probes labeled with marker: radioisotope,
fluorochrome, enzyme or chemiluminescent
substrate.
Hybridization can take place in solid support
medium or liquid.
Solid Support Hybrization
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Dot-blot
Sandwich hybridization
Gel electrophoresis
Southern Blot
Northern Blot
Dot-Blot
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Dot-blot clinical sample applied to membrane,
heated to denature DNA.
Labeled probes added and will bind to target
if present.
Wash to remove unhybridized probe.
Detect by autoradiography or enzyme assay.
Qualitative test only.
May be difficult to interpret.
Dot-Blot Hybridization
Sandwich Hybridization
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Uses two probes, one bound to membrane and serves
as capture target for sample DNA of interest.
Second probe anneals to different site on target DNA
and has label for detection.
Sample nucleic acid sandwiched between the two.
Two hybridization events occur, increases
specificity.
Can be adapted to microtiter plates.
Sandwich Hybridization
Characterization of DNA
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Restriction endonucleases cleave DNA at
SPECIFIC recognition sites approximately 4
to 6 base pairs long.
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Enzyme which cuts ds or ss DNA at specific site.
Enzymes found naturally.
Over 3000 identified, 500 available
commercially.
Human DNA can yield millions of unique
fragments.
Characterization of DNA
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EcoRI popular restriction endonuclease.
Cuts wherever there is a G-C or C-G bond.
Fragments separated based on size and electrical charge by
electrophoresis.
Kits developed to detect specific gene fragments associated
with diseases or conditions
Characterization of DNA
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Differences in restriction patterns referred to
as restriction fragment length polymorphisms
(RFLP).
Caused by variations in nucleotides within
genes that change where restriction enzymes
cleave DNA.
When mutations occur different sized pieces
of DNA are obtained.
Use of RFLP
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Identify specific microorganisms
Detect polymorphism in MHC genes.
DNA fingerprinting – first method used
Localization of genes for genetic disorders
Determination of risk for disease
Paternity testing
Use of RFLP
Southern Blot
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Used to detect a specific DNA sequence in a
sample.
Use restriction endonuclease to cut DNA into
fragments.
DNA fragments separated by electrophoresis.
Electrophoresis to Separate Fragments
Southern Blot
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Pieces denatured and transferred from gel to membrane for
hybridization reaction.
Place membrane on top of gel, add weights or use suction and
allow buffer plus DNA to wick up onto the membrane.
Once DNA is on membrane heat or use UV light to crosslink
strands to permanently attach the DNA onto membrane.
Add labeled DNA probes for hybridization to take place.
Radioactive, fluorescent or chromogenic dye used as label
Probes added in excess so target molecules reanneal and more
likely to attach to probe.
Visualize results based on label used.
Southern Blot
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Southern Blot takes advantage of the fact that DNA fragments will stick to a nylon or
nitrocellulose membrane.
Membrane laid on top of the agarose gel and absorbent material (e.g. paper towels or a
sponge) is placed on top.
With time, the DNA fragments will travel from the gel to the membrane by capillary action
as surrounding liquid is drawn up to the absorbent material on top.
After the transfer of DNA fragments has occurred, the membrane is washed, then the DNA
fragments are permanently fixed to the membrane by heating or exposing it to UV light.
The membrane is now a mirror image of the agarose gel.
Southern Blot
Southern Blot
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http://www.koreanbio.org/Biocourse/index.php/Hybridization
Southern Blot
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MOM [blue], DAD [yellow], and their four children: D1 (the
biological daughter), D2 (step-daughter, child of Mom and
her former husband [red]), S1 (biological son), and S2
(adopted son, not biologically related [his parents are light
and dark green]).
Northern Blot
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Northern blots used to detect RNA in a sample.
RNA (either total RNA or just mRNA) separated by gel electrophoresis.
RNA transferred to sheet of nitrocellulose, though other membranes can
be used and immobilized by heat or UV light.
Blot is incubated with a probe which is single-stranded DNA.
This probe will form base pairs with its complementary RNA sequence
and bind to form a double-stranded RNA-DNA molecule. The probe
cannot be seen but it is either radioactive or has an enzyme bound to it
(e.g. alkaline phosphatase or horseradish peroxidase).
Location of the probe is revealed by incubating with a substrate that the
attached enzyme converts to a colored product that can be seen or gives
off light when exposed on X-ray film.
If the probe was labeled with radioactivity, it can expose X-ray film
directly.
Northern Blot
Solution Hybridization
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Both target nucleic acid and probe free to interact in
solution.
Hybridization of probe to target in solution is more
sensitive than hybridization on solid support
Requires less sample and is more sensitive.
Probe must be single-stranded and incapable of selfannealing.
Fairly adaptable to automation, especially those
using chemiluminescent labels.
Assays performed in a few hours.
Solution Hybridization
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Second method is hybridization protection assay.
Chemiluminescent acridinium ester attached to
probe as label.
After hybridization solution subjected to alkaline
hydrolysis.
Causes hydrolysis of ester if probe not attached to
target.
If probe attaches to target light given off.
Solution Hybridization
In-Situ Hybridization
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Target nucleic acid found in intact cells.
Provides information about presence of
specific DNA targets and distribution in
tissues.
Probes must be small enough to reach nucleic
acid.
Radioactive or fluorescent tags used.
Requires experience.
In-Situ Hybridization
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Fluorescent in-situ hybridization (FISH) very
popular.
Cytogenetic technique used to detect and
localize presence or absence of DNA
sequences on chromosomes.
Used in genetic counseling, medicine and
species identification.
FISH
FISH
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A metaphase cell positive for the bcr/abl rearrangement
(associated with chronic myelogenous leukemia) using FISH.
The chromosomes can be seen in blue. The chromosome that
is labeled with green and red spots (upper left) is the one
where the wrong rearrangement is present.
FISH
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DNA probes specific to regions of particular chromosomes are attached to
fluorescent markers and hybridized with a chromosome spread.
Picture shows a computer-generated "false colour" image, in which small
variations in fluorescence wavelength among probes are enhanced as distinct
primary colours.
The combination of probes that hybridize to a particular chromosome produces a
unique pattern for each chromosome. This makes it particularly easy to detect
segmental deletions and translocations among chromosomes.
DNA Chip aka Microarrays
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A DNA chip (DNA microarray) is a biosensor which analyzes gene
information from humans and bacteria.
Utilizes the complementation of the four bases labeled A, T, G and C in
which A pairs with T and G pairs with C through hydrogen bonding.
Solution of DNA sequences containing known genes (DNA probe) placed
on glass plates in microspots several microns in diameter arranged in
multiple rows.
Genes are extracted from samples such as blood, amplified and then
reflected in the DNA chip, enabling characteristics such as the presence
and mutation of genes in the test subject to be determined.
As gene analysis advances, the field is gaining attention particularly in the
clinical diagnosis of infectious disease, cancer and other maladies.
Microarrays
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Microarrays are a significant advance both because they may
contain a very large number of genes and because of their
small size.
Useful in surveying large number of genes quickly or when
the sample to be studied is small.
May be used to assay gene expression within a single sample
or to compare gene expression in two different cell types or
tissue samples, such as in healthy and diseased tissue.
Because a microarray can be used to examine the expression
of hundreds or thousands of genes at once, it promises to
revolutionize the way scientists examine gene expression.
This technology is still considered to be in its infancy.
Microarray
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Fix the single stranded DNA to chip.
Labeled with different colors.
Microarray
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A microarray scanner provides a picture of what spots showed up green,
red, or yellow.
A green dot would represent a gene that is expressed more in healthy cells
or have less expression in cancer cells, and a red dot represents a gene that
is expressed more in cancer cells.
A yellow spot would be a gene that is expressed in both cancer and
healthy cells.
Microarray
Drawbacks
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Stringency, or correct pairing, is affected by:
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Salt concentration
Temperature
Concentration of destabilizing agent such as formamide or
urea.
If conditions not carefully controlled mismatches can
occur.
Patient nucleic acid may be present in small
amounts, below threshold for probe detection.
Sensitivity can be increased by amplification: target,
probe and signal
DNA Sequencing
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Method to determine the exact order of the
nucleotide bases in DNA.
Unknown DNA sequences compared to
known.
Several methods available.
Sanger method of choice.
Sanger Method of DNA Sequencing
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Requires ss DNA template, DNA primer, DNA polymerase,
labeled nucleotides and modified nucleotides to terminate
DNA elongation.
DNA sample divided into 4 separate reactions to normal
(NTP) and ONE type of dideoxynucleotides (ddNTP) A, T, C
or G are added.
ddNTPs will prevent addition of further nucleotides.
Creates DNA strands of discrete sizes.
Each reaction loaded on separate lane on gel and
electrophoresed.
Sequence of nucleotides read in order to determine DNA
sequence.
DNA Sequencing
Sanger’s DNA Sequencing
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Excellent explanation with animations – MUST see.
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http://www.dnalc.org/view/15922-Early-DNA-sequencing.html
Target Amplification
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In-vitro systems for enzymatic replication of target
molecule to detectable levels.
Allows target to be identified and further
characterized.
Examples:
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Polymerase chain reaction (PCR)
Transcription mediated amplification (TMA)
Strand displacement amplification (SDA)
Nucleic acid sequence-based amplification (NASBA)
Polymerase Chain Reaction
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Capable of amplifying tiny quantities of nucleic acid.
Cells separated and lysed.
Double stranded DNA separated into single strands (denatured).
Primers, small segments of DNA no more than 20-30 nucleotides long, added.
Primers are complementary to segments of opposite strands of that flank the target
sequence.
Only the segments of target DNA between the primers will be replicated.
Each cycle of PCR consists of three cycles:
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Denaturation of target DNA to separate 2 strands by heating.
Annealing step in which the reaction mix is cooled to allow primers to anneal to target
sequence
Extension reaction in which primers initiate DNA synthesis using a DNA polymerase.
These three steps constitute ONE thermal cycle
Each PCR cycle results in a doubling of target sequences and typically allowed to
run through 30 cycles, one cylce takes approximately 60-90 seconds.
Taq
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Taq polymerase ("Taq pol") is a thermostable
polymerase isolated from thermus aquaticus, a
bacterium that lives in hot springs and
hydrothermal vents.
"Taq polymerase" is an abbreviation of
Thermus Aquaticus Polymerase.
Often used in polymerase chain reaction,
since it is reasonably cheap and it can survive
PCR conditions.
Polymerase Chain Reaction
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http://www.dnalc.org/resources/animations/pcr.html
Polymerase Chain Reaction
Polymerase Chain Reaction
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Sample with all test
components mixed
together.
Put in thermocycler
which cycles the
temperature for each
stage of the reaction.
Electrophorese to
separate and identify.
Transcription Mediated Amplification
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Two Types to discuss:
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Nucleic acid sequence-based amplification
(NASBA)
Transcription mediated amplification (TMA)
Nucleic Acid Sequence-Based
Amplification (NASBA)
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Alternative amplification procedure.
One step isothermal process for amplifying
RNA.
NASBA has proven to be successful in
detection of both viral and bacterial RNA in
clinical samples.
NASBA
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The NASBA technique has been used to develop
rapid diagnostic tests for several pathogenic viruses
with single-stranded RNA genomes:
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influenza A
foot-and-mouth disease virus
severe acute respiratory syndrome (SARS)-associated
coronavirus
Human bocavirus (HBoV)
Parasites like Trypanosoma brucei
Transcription Mediated Amplification
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Another alternate amplification method.
TMA is an RNA transcription amplification system using two enzymes to
drive the reaction: RNA polymerase and reverse transcriptase.
TMA is isothermal; the entire reaction is performed at the same
temperature in a water bath or heat block. This is in contrast to other
amplification reactions such as PCR or LCR that require a thermal cycler
instrument to rapidly change the temperature to drive the reaction.
TMA can amplify either DNA or RNA, and produces RNA amplicon, in
contrast to most other nucleic acid amplification methods that only
produce DNA.
TMA has very rapid kinetics resulting in a billion fold amplification
within 15-30 minutes.
TMA
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Tests have been developed for:
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Chlamydia trachomatis
Neisseria gonorrhoeae
Mycobacterium tuberculosis
Mycobacterium avium
HIV-1
HBV
HCV
Strand Displacement Amplification
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An isothermal process that permits 1010-fold
amplification of a DNA target sequence in as
little as 15 min.
Method for producing abundant DNA from
valuable and often limited clinical specimens
Isothermal nature of the reaction process
offers distinct advantages with regard to cost
and simplicity of instrumentation.
Universal detection format permits the use of
the same fluorescent detector probes across
multiple analytes.
Strand Displacement Amplification
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Used for detection of:
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Neisseria gonorrhoea
Chlamydia trachomatis
Mycoplasma pneumoniae
Mycobacteria tuberculosis
Ligase Chain Reaction
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Method of DNA amplification.
Amplifies the nucleic acid used as a probe.
Two probes are used per each DNA strand and are
ligated together to form a single probe.
Uses both a DNA polymerase enzyme and a DNA
ligase enzyme to drive the reaction.
Requires thermocycler.
Each cycle results in doubling of target.
Greater specificity than PCR.
Ligase Chain Reaction
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Used for
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Detection of single base mutations in genetic
diseases.
Neisseria gonorrhoea
Chlamydia trachomatis
Drawbacks of Amplification Systems
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Potential for false-positive results due to contaminating
nucleic acids.
PCR and LCR, DNA products main source of contamination.
TMA, RNA products are possible contaminants.
Must have product inactivation as part of QC program.
Separate preparation areas from amplification areas and use
of inactivation systems such as UV light help alleviate
contamination.
Very expensive.
Closed system, automation will also decrease number of
problems.
Future of Molecular Diagnostic Techniques
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Despite expense may be times that rapid diagnosis will result in decreased
cost.
Example: Mycobacteria - quick diagnosis no need for expensive
respiratory isolation.
Detection of multi-drug resistant M. Tuberculosis will lead to more timely
public health measures.
Incredibly useful in serology and microbiology.
Increased specificity and sensitivity of molecular testing will become the
standard of practice in immunology and microbiology.
Testing will continue to become more rapid as assays are automated
which will also bring down the costs.
Author states will not replace culture for routine organisms, but it already
is, and as DNA chip technology improves, the ability to test for multiple
organisms will become easier
References
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http://www.brc.dcs.gla.ac.uk/~drg/courses/bioinformatics_mscIT/slides/slides2/sld001.htm
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http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GelBlotting.html
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http://www.bioteach.ubc.ca/MolecularBiology/IdentifyingDNA/
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http://www.bio.davidson.edu/courses/genomics/Front/surfingenomics.html
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http://ccm.ucdavis.edu/cpl/Tech%20updates/TechUpdates.htm

http://www.cdc.gov/ncidod/eid/vol7no2/pfaller.htm