DNA Analysis
PCR
Polymerase Chain Reaction
is an important technique in molecular genetics that permits the
analysis of short sequences of DNA (or RNA). Think
of it as a means to photocopy (amplify) short stretches of DNA or
RNA. Previously, amplifying was done in bacteria, and took weeks. But
now, with PCR carried out in single-tube reactions, it takes only a
few hours. PCR is so highly efficient that untold numbers of copies
can be made of the DNA.
To copy DNA by PCR, you need the same molecules that nature uses
for copying DNA:
- Two primers that flag the beginning and end of the DNA stretch
to be copied;
- An enzyme called polymerase that walks along the segment of DNA,
reading its code and assembling a copy;
- DNA building blocks that
the polymerase needs to make that copy.
As
illustrated in the animation, three major steps are involved
in a PCR. Each step takes
place at a different temperature. (click on the link, the animation
will open in a new browser window).
- Denaturation: At around 94°C, the double-stranded DNA melts
and opens into single-stranded DNA. You have to have single-stranded
DNA to make a copy—it becomes the template.
- Annealing: The temperature at this step
depends on the sequence of the DNA to be copied or amplified—around
54°C.
It is a temperature that allows the primers to bind the
DNA, but
is not cool
enough to permit the two DNA strands to come together.
Once the primers bind, DNA polymerase (the copying enzyme) is
able to
attach to the
primers.
- Extension: At 72°C, the polymerase works
best and begins copying the DNA. The result is a double stranded
region of DNA complementary
to the template sequence. In order to make another copy,
the double-stranded DNA must be denatured—and we’re
back to step 1.
These three steps can be cycled many times
(usually around 30 times) in a thermocycler, which is an
instrument that
is able
to carefully
regulate the temperature in a cyclical pattern.
DNA Sequencing
 DNA
sequencing is the process used to read the nucleotide sequence
of a region of DNA. We utilize automated fluorescent dye-terminator
cycle sequencing, based on the chain-termination dideoxynucleotide
method. It incorporates dideoxynucleotide-conjugated fluorescent
dyes in a PCR-based primer extension sequencing reaction. Each
nucleotide is attached to a unique fluorescent dye. Thus, the
identity of the dye corresponds to the final base on that fragment.
The entire reaction is purified, then run in a single lane on
a polyacrylamide gel so that the fragments separate according
to size. As the fragments migrate on the gel, they run past a
laser detector, and the emission wavelength of each fragment
is detected. The result is a chromatogram corresponding to the
DNA sequence.
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Example of a sequencing chromatogram
generated from the preceding gel.
The A, T, G, C letters are the DNA sequence.
SNP Genotyping
Just as fingerprints are unique to a person, each
person’s
genome is slightly different. These individual genetic
variations are known
as polymorphisms. The most common type of polymorphism
is a single nucleotide polymorphism (SNP). These single nucleotide
variations
occur about once every 1,000 bases. SNPs are very useful
in searching for genes
related to multi-factorial diseases or developmental
conditions. We utilize the ABI SNP allelic discrimination assay
based upon the TaqMan
method (see quantitative
real time RT-PCR). The result
is a graph
wherein each dot represents a sample of DNA. The
dots are grouped
by the type of SNP detected. We are using these tests to look for
genes and mutations linked to vocal disorders and a
tender larynx condition.
Example of SNP Genotyping Data
Denaturing High Performance Liquid Chromatography
(DHPLC)
DHPLC identifies mutations without
sequencing. It is a cost-effective method to screen large numbers
of crude PCR products for
mutations and polymorphisms. Direct sequence analysis
can
be used to
confirm DHPLC results. This method efficiently
detects single nucleotide
and insertion/deletion variations in PCR products
directly without purification.
DHPLC Principle
DHPLC identifies mutations
and polymorphisms based on detection of heteroduplex formation
between mismatched
nucleotides
in PCR amplified DNA. Sequence variation creates
a mixed population of heteroduplexes and homoduplexes during
reannealing of
wild type and
mutant DNA.
DHPLC uses an ion-pair reversed-phase
high performance liquid chromatography method (IP-RP-HPLC). DNA is
mixed with an
ion-pairing agent of
triethylammonium acetate (TEAA). This mix is passed
through a column comprised of
a polystyrene-divinylbenzene copolymer which binds
the DNA. A linear gradient of acetonitrile allows
separation of fragments
based on
size
and/or presence of heteroduplexes. When this mixed
population
is analyzed by HPLC under partially denaturing
temperatures, the heteroduplexes
elute from the column earlier than the homoduplexes
because of
their reduced melting temperature. As the fragments
elute, they are UV detected (260 nm). Analysis can be performed on
individual samples to determine heterozygosity, or on mixed
samples to identify sequence variation between individuals.
Sample DHPLC Data. Arrow
shows the mutation (bottom left)
in the sequence that results in the Heteroduplex DHPLC (bottom
right).
The normal is shown on the top row.
Luciferase
Reporter Gene Assay
Promoters are regions of DNA near the start
of a gene which influence the expression of that gene. Promoters,
along
with suppressors
and enhancers, play an important role in the
molecular response of a
cell. Reporter genes have been used to study
these regulatory pieces of DNA.
Reporter genes are nucleic acid sequences encoding
easily assayed genes such as the firefly Luciferase
gene which
oxidizes luciferin,
creating
light. Reporter genes can be attached to regulatory
regions of DNA to see if that regulatory piece
of DNA is affected
by a given
condition
(e.g. vibration as in the vocal folds of the
lamina propria). We are using various Luciferase reporter
gene assays
to examine the
regulatory
elements of genes differentially expressed in
the larynx.
Luciferase Reaction
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