Experiment Discussion Description: Taq DNA Polymerase Chain Reaction

Running head: EXPERIMENT DISCUSSION
Experiment Discussion
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1EXPERIMENT DISCUSSION
Introduction
The purpose of polymerase chain reaction lies in identifying the mutations leading to
genetic disorders. This powerful method involve amplification of specific DNA sequences
resulting in exponentially increased target DNA sequences. The PCR techniques involves the
use of Taq DNA polymerase which uses a template DNA to synthesize deoxynucleotides.
The polymerase performs adding of new nucleotides to the 3’ end of a primer sequence and
extends this sequence in an exponential manner (Garibyan and Avashia 2013). The primer is
designed such that it has a region of complementarity to the target DNA sequence, this region
contains the mutations or polymorphism which is amplified by PCR (Rahman et al. 2013).
This amplified DNA sequence is treated with a suitable restriction enzyme which
recognizes the polymorphic sequence and produces the respective DNA fragments after
restriction digestion, a process termed as restriction fragment length polymorphism (RFLP).
Polymorphic site within the amplified DNA sequence digested by restriction enzyme
produces different lengths of digested fragments which are observed by performing
polyacrylamide gel electrophoresis. The restriction digestion reaction involves the DNA
template, restriction enzyme and a buffer solution to perform the restriction. These enzymes
are restriction endonucleases which recognize the particular sequence and cut within that
recognition sequence to give rise to digestion fragments.
The PCR amplification of DNA sequence is visualized through gel electrophoresis.
Gel electrophoresis clearly shows the amplified DNA bands which are visualized using
ethidium bromide intercalating between DNA, visible under ultraviolet light (Mesapogu et al.
2013). The aim of the study therefore is to investigate the genetic variations that give rise to
genetic diseases. The PCR followed by RFLP and gel electrophoresis indicates the
identification of single nucleotide polymorphism (SNP) that is found to be associated with

2EXPERIMENT DISCUSSION
genetic disease pathogenicity (Anderson et al.2013). These genetic test are performed to
confirm the likelihood of inheritance of polymorphism to determine the susceptibility of
genetic disease among the next generation.
Results
The pcr technique was conducted to amplify the target DNA sequence which is suspected to
contain the single nucleotide polymorphism. The amplified pcr products are treated with
restriction enzyme which determines the presence of disease or normal condition. This
amplification was visualised through gel electrophoresis under ultraviolet light.
The results of PCR is suggestive of the fact that many positive amplification is not obtained
as expected from the backup pcr image. The gel image is not clear for observation, however
result information can be obtained through deduction. There may be handling errors while
making pcr samples and transferring the right volumes, therefore the chain reaction may not
have occurred as expected. Moreover, the volume and gel concentration may be leading to
amplification errors.
From the obtained results, it can be said that band 2 shows dominant homozygous condition;
band 3 shows homozygous recessive condition ; band 4 is a carrier of the disease.
Discussion
To perform accurate experimental design and therefore accuracy in results, control
samples are taken. Here, 2 negative controls, one with DNA+ restriction enzyme and the
other with DNA only are used during RFLP gel run. Negative controls should not give any
band in the gel, which is indicative of the fact that the experiment performed is sterile and
accurate. Presence of any band for negative control samples signifies that there is presence of
contamination in reagents or an issue with sterile handling (Kozera and Rapacz 2013).