The Polymerase Chain Reaction (PCR) is a technique used to artificially
reproduce chains of deoxyribose nucleic acid (DNA) strands by amplifying “copies” of a
target segment of DNA over and over again until a maximum amount of the target DNA
has been produced. Through the PCR process, long segments of a known or unknown
sequence can be duplicated. While amplification techniques are important, genotyping
specific sequences of a DNA strand is useful in screening for diseases or mutations and
can also determining organ transplant compatibility in time for a patient to survive.
Once the PCR process has generated enough DNA for genotyping scientists can
then identify a DNA segment as a specific sequence among a selection of sequences.
PCR is controlled by distinct temperature changes known as a thermocycle. The
temperature of the mixture is increased to denature the target strand of DNA, then the
mixture is cooled and primers (a catalyst) anneal to each of the complimentary strands of
the target DNA. Finally amplification of the strand occurs either at an intermediate
temperature or while the temperature is brought back to its highest level in order to
denature the DNA strands again, thus repeating the PCR process. Once a maximum
amount of DNA is artificially reproduced, the sample is heated slowly one final time to
allow for genotyping. This slow melt process releases fluorescent dye upon the DNA
uncoiling, which measures how much DNA is wound or unwound and generates a
fluorescence curve used for genotyping. The success and efficiency of the PCR process
depends on the primers used and the temperature cycling conditions.
The temperature levels for various DNA strands depend on the specific sequence,
and scientists are beginning to approximate melting temperatures for various known
segments of DNA. High resolution melting curve analysis is now used by scientists to
quantify and identify specific product sequence duplicated by PCR. Amounts of the
amplified product are usually doubled each cycle and grow exponentially in the
beginning of the PCR reaction. Eventually the total product amount levels off to a steady
amount (a flat plateau graphically) before dropping slightly. The amount of amplified
DNA is calculated by the total fluorescence radiated during the PCR process. As primers
anneal correctly to and extend along the amplicon (DNA to be amplified), a dye binds
between the DNA strands and fluoresces. As more product is amplified and coiled
correctly, more fluorescent dye will be detected.
As an REU student, I will be working with Bob Palais on genotyping DNA by
high-resolution DNA melting curve analysis, specifically looking at dynamic melting
temperatures and curve models involving statistical mechanics and thermodynamics.
These methods involve multivariable calculus, linear algebra, and differential equations.
By incorporating the effect of slow vs. fast melting temperatures and the presence of
fluorescent dyes, we will able to improve our ability to genotype specific DNA strands.
Base specificity is also a key issue worthy of research. There are four bases which
are the building blocks of DNA: A, T, G, C. The way that these bases pair together
determine whether there are mutations in the amplified DNA or whether the DNA
sequence is amplified correctly. A’s pair up with T’s and G’s pair with C’s. If the wrong
bases pair together, the fluorescent dye will not fluoresce, or in some cases, the wrong
color of dye will fluoresce. We will compare the melting temperature and fluorescence of
AT pairs compared to GC pairs, and also compare how much dye will fluoresce when
there is a mutation present or absent in the DNA sequence. Base specificity affects
melting curves and the quantification of how much coiled DNA is present. Bob and I will
be focusing on base specificity and its effects on melting temperatures and fluorescence
as well as general genotyping of specific DNA sequences.