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Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
Published on: November 10, 2015
Decreasing amplification bias associated with multiple displacement amplification and short tandem repeat genotyping
Kaye N Ballantyne1, Roland A H van Oorschot, Iman Muharam
1Biology Division, Victoria Police Forensic Services Department, Macleod, 3085 VIC, Australia. kaye.ballantyne@police.vic.gov.au
Analytical Biochemistry
|June 23, 2007
Summary
Multiple displacement amplification (MDA) bias affects genotyping, especially with small DNA amounts. Omitting template denaturation reduces this bias, and combining both denaturation methods further minimizes errors in SNP and STR analysis.
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- Multiple displacement amplification (MDA) is a widely used whole genome amplification technique.
- Amplification bias from varphi29 polymerase can impact genotyping accuracy, particularly for single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) with low template DNA.
- Bias can manifest between different genomic loci or between alleles within the same locus.
Purpose of the Study:
- To investigate the impact of template denaturation on MDA-induced amplification bias.
- To evaluate strategies for mitigating bias in SNP and STR genotyping.
- To assess the utility of combining denatured and nondenatured MDA reactions for improved genotyping and copy number analysis.
Main Methods:
- Comparing MDA with and without template denaturation.
- Analyzing bias in SNP and STR genotyping across different loci and alleles.
- Implementing a dual MDA approach combining both denaturation methods.
Main Results:
- Omitting template denaturation significantly reduces MDA amplification bias.
- Nondenaturation reverses the allelic bias direction observed in standard MDA.
- Combining denatured and nondenatured MDA reactions substantially reduces bias, enhancing copy number analysis and genotyping accuracy.
Conclusions:
- Template denaturation is a critical factor influencing MDA bias in genotyping.
- A dual MDA approach offers a robust strategy to minimize amplification bias.
- This method improves the reliability of genotyping and copy number analysis, especially in low-input DNA samples.
