Whole genome amplification of single cells: mathematical analysis of PEP and tagged PCR

Abstract

We construct a mathematical model for two whole genome ampliffication strtegies, prinwr extension pmampiffication (PEP) and tagged Fpoly aee chain reaction (tagged PCR). An explicit formula for the expected target yield of PEP is obtained. The distribution of the target yield and the coverage prties of these two stralegies are studied by simultions. From our studies we find that polymerase wth high processivity may increase the efficiency of PEP and tagged PCR.

Genetic analysis using genomic representations

“…The usefulness of the resulting PCR products for genomic analysis was not explored; nor was its efficiency explored when starting with tiny amounts of material. An alternate approach to whole genome PCR is to use random priming with degenerate oligonucleotides (29)(30)(31)(32)(33). This method can produce large amounts of DNA starting from very minute amounts of sample, but the complexity of the DNA produced, the reproducibility of the representation, and the loss of natural restriction sites at the ends of the amplified material make the usefulness of this method somewhat limited.…”

“…After transfer, the blot was hybridized in GIBCO prehybridization solution at 67°C for 2 hr. The probe was produced by random priming with the rediprime kit in the presence of 32 P␣dCTP. After 12-18 hr of hybridization, the blots were washed with standard saline phosphate͞EDTA buffer as described (6).…”

“…LOH was assessed by using the tetranucleotide repeat marker D17S695 (listed as UT269 in the National Center for Biotechnology Information Web Site http:͞͞ www.ncbi.nlm.nih.gov͞Entrez͞nucleotide.html), which maps to the 17p13 region of the human genome and has been used to determine the state of LOH at the p53 locus (7,8). The reverse primer was labeled by phosphorylation in the presence of 32 P␥ATP as described (7,8). The labeled reverse primer in combination with both forward and reverse primers was used for PCR by using 50 ng of HCR as DNA template.…”

Whole Genome Amplification of DNA from Laser Capture-Microdissected Tissue for High-Throughput Single Nucleotide Polymorphism and Short Tandem Repeat Genotyping

“…23 Theoretical calculations have also predicted that tagged PCR cannot provide complete genomic coverage at low cell numbers. 24 Because MDA uses completely randomized primers, broader genomic coverage and less locus bias were achieved. 20 A performance comparison between DOP-PCR and pooled MDA for LCM cells is shown in Table 3, based on our results and that from a widely cited reference.…”

“…24 In practice, PEP typically generates only a moderate ϳ30-fold amplification with a ϳ100-fold locus bias. 20 A recent report demonstrated that STR analysis on an ABI sequencer was achieved using PEP products from 5000 LCM cells (Table 3).…”