Focus: Amplification

GoTaq® Hot Start Polymerase Successfully Amplified Allele C of Polymorphism 311 (CysSer) on the PON2 Gene

We compared GoTaq® Hot Start Polymerase with the GoTaq® Flexi DNA Polymerase when used to amplify allele C of polymorphism 311 (Cys→Ser) on the PON2 Gene. GoTaq® Hot Start DNA Polymerase amplified the 96bp product without nonspecific bands.

By Gerardo Pérez, M.Sc., Lizbeth González, Ph.D., David Oaxaca, Ph.D., Diana Franco, B.S. and Ariana Vargas, B.S.
Universidad Autónoma de Yucatán
Departamento de Salud Reproductiva y Genetica (SAREG) del Centro de Investigaciones Regionales
Ciudad de Mérida, Yucatán, México.

Published in July 2008

Introduction

The gene of an esterase enzyme, called paraoxonase (PON), is a member of a multigene family that comprises three related genes (PON1, PON2 and PON3) with structural homology clustering on the chromosome 7 (1,2). Polymorphisms of PON2 gene are associated with risk of cardiovascular diseases such as hypercholesterolemia, noninsulin-dependent diabetes, coronary heart disease (CHD) and myocardial infarction. The codon 192 mutation of PON1 gene is associated with the risk of CHD and has a synergistic effect with the codon 311 (Cys→Ser) polymorphism of PON2 gene. Moreover, the PON2 codon 311 (Cys→Ser) polymorphism might interact with the apoE4 allele to increase the risk of dementia (3).

The polymorphism 311 (Cys→Ser) of PON2 gene was determined by allele-specific oligonucleotide PCR assay (ASO-PCR) using primers described by Chen et al. (4) with patient DNA. This PCR method showed a 96bp DNA fragment for allele C and a 104bp DNA fragment for allele S. In this report, we show the genotyping of allele C in five subjects using both GoTaq® Flexi DNA Polymerase and GoTaq® Hot Start Polymerase (Cat.# M5001).

Materials and Methods

The allele C of polymorphism 311 (Cys→Ser) on PON2 gene was determined by ASO-PCR using the primers in reference 4 with either GoTaq® Flexi DNA Polymerase or GoTaq® Hot Start Polymerase in a 25µl volume. See Table 1 for reaction component volumes and Table 2 for cycling conditions.

Table 1. PCR Mix.
Component Volume
5X Green GoTaq® Flexi Buffer 5μl
25mM MgCl2 1μl
10mM dNTP mix 0.5μl
20pM upstream primer 0.3μl
20pM downstream primer 0.3μl
5u/µl GoTaq® Flexi DNA Polymerase or
GoTaq® Hot Start Polymerase		 0.13μl
100ng template DNA 1µl
nuclease-free water to 25μl
Table 2. Cycling Conditions.
Temperature (°C) Time Cycles
Initial Denaturation 94 3 minutes 1
Denaturation 94 30 seconds 35
Annealing 60 30 seconds
Extension 72 30 seconds
Final Extension 72 10 minutes 1

PCR products were resolved by electrophoresis on a 10% polyacrylamide (Cat.# V3111) gel , and stained with silver nitrate.

Results

thumbnail-Genotyping of allele C of polymorphism 311 (Cys→Ser) on PON2 gene
Genotyping of allele C of polymorphism 311 (Cys→Ser) on PON2 gene

Figure 1. Genotyping the 96bp PCR product of allele C, polymorphism 311 (Cys→Ser) on the PON2 gene. Panel A. The 96bp product of allele C of polymorphism 311 (Cys→Ser) on PON2 gene was amplified using GoTaq® Flexi DNA Polymerase and separated by polyacrylamide gel electrophoresis. Note the nonspecific bands obtained along with the DNA fragment of expected size (area highlighted by brackets). Lane M, 100bp DNA Ladder (Cat.# G2101); lanes 1–5, allele C amplimers; and lane 6, negative control (no DNA). Panel B. The 96bp amplimer of allele C of polymorphism 311 (Cys→Ser) on PON2 gene generated using the GoTaq® Hot Start Polymerase (Promega). In contrast to the brackets in Panel A, there are no nonspecific products present. Lane M, 100bp DNA Ladder (Cat.# G2101); lanes 1–5, allele C amplimer; and lane 6, negative control (no DNA).

Conclusions

We obtained more specific and cleaner PCR products for allele C of polymorphism 311 (Cys→Ser) on PON2 gene using GoTaq® Hot Start Polymerase compared to GoTaq® Flexi DNA Polymerase, eliminating nonspecific products. GoTaq® Hot Start Polymerase improved our PCR determination of the polymorphism C311S, offering significant advantages by increasing specificity and improving amplification effectiveness.

References

  1. Sorenson, R. et al. (1995) The genetic mapping and gene structure of mouse paraoxonase/arylesterase. Genomics 30, 431–8.
  2. Primo-Parmo, S. et al. (1996) The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family. Genomics 382, 498–507.
  3. Janka, Z. et al. (2002) Codon 311 (Cys→Ser) polymorphism of paraoxonase-2 gene is associated with apolipoprotein E4 allele in both Alzheimer’s and vascular dementias. Molecular Psychiatry 7, 110–2.
  4. Chen, J. et al. (2003) Increased influence variation on PON1 activity in neonates. Enviro. Health Perspectives 111 (11), 1403–09.