Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping.
Multiple displacement amplification (MDA) can generate large quantities of genomic DNA product from small amounts of template. We investigated the ability of MDA to amplify samples containing very small amounts of target DNA (5 pg to 1 ng) in the presence of a second, larger DNA sample for downstream short tandem repeat (STR) multiplex genotyping. We observed that STR amplification success of the minor fraction was increased in these mixed samples when compared with standard PCR only or MDA containing only the single trace DNA sample. Increased numbers of alleles were detected, with less amplification bias between loci than in single source samples undergoing the same protocol. To improve the STR genotyping accuracy, animal DNA was substituted for the additional human DNA, maintaining the increase in the number and quality of human-specific STR loci amplified. Polyethylene glycol 400, a commonly used crowding agent, was used as a replacement for the added genomic DNA in the MDA reaction and produced very similar results. Therefore, we suggest that additional DNA is acting as a molecular crowding agent during MDA. Performing MDA on trace amounts of DNA under crowded conditions results in greater numbers of alleles being amplified and more balanced amplification occurring between alleles.
Ballantyne
Kaye N
KN
Biology Division, Victoria Police Forensic Services Department, Macleod, VIC 3085, Australia. kaye.ballantyne@police.vic.gov.au
van Oorschot
Roland A H
RA
Mitchell
R John
RJ
Koukoulas
Irene
I
D000483:Alleles; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D005819:Genetic Markers; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences
D005819:Genetic Markers; D004247:DNA