Real-time PCR was performed on 47 human loci (2 loci on each autosomal pair, 2 loci on the X chromosome(s), and 1 locus on the Y chromosome) from 44 different samples amplified using REPLI-g technology. Each sample was amplified approximately 10,000-fold with a maximum bias of representation between the loci being only 6-fold.
Various amounts of human genomic DNA were amplified in a standard REPLI-g Midi Kit reaction and aliquots taken at the indicated timepoints. The yield of amplified DNA from a 50 µl reaction was approximately 40 µg, regardless of the amount of starting material.
DNA amplified using REPLI-g technology, without subsequent purification, was subjected to SNP genotyping at 2 randomly selected loci (WIAF-1004 and WIAF-622) using TaqMan® analysis. Tight clusters of alleles allow reliable determination of genotyping of homo- and heterozygote genotypes.
Real-time PCR of REPLI-g amplified DNA samples stored in 4 different formats at –20°C for the indicated time periods. Two loci, [A] locus A and [B] locus B, were assayed for each sample. gDNA: genomic DNA not amplified with REPLI-g. Storage formats: 50 µl REPLI-g reactions: 1) without further manipulation ("50 µl REPLI-g"); 2) aliquoted to 5 µl volumes ("5 µl REPLI-g"); 3) purified with QIAamp Mini Kit ("50 µl QIAamp purified REPLI-g"); and 4) diluted to a concentration of 50 ng/µl (50 µl diluted REPLI-g").
Genomic DNA samples (10 ng) were denatured using heat (95°C) or the standard REPLI-g Kit alkaline lysis protocol. After amplification using REPLI-g DNA Polymerase the CT values of 2 loci were compared between samples. The low CT values of loci amplified using the REPLI-g Kit alkaline lysis protocol indicate better locus representation, meaning there has been no loss of sequence information at these loci.
The relative representation of 8 loci was determined using real-time quantitative PCR for DNA amplified using [A] REPLI-g technology [B] DOP-PCR and [C] PEP. Locus representation was determined by comparison to 1 µg of unamplified control DNA.
DNA polymerase moves along the DNA template strand displacing the complementary strand. The displaced strand becomes a template for replication, enabling high yields of high-molecular-weight DNA to be generated.
Amplification of genomic DNA using the REPLI-g Mini Kit involves 3 basic steps. First, the sample (10 ng purified genomic DNA, 0.5 µl whole blood or tissue culture cells) undergoes gentle alkaline denaturation, avoiding fragmentation and damage of template DNA. Next the sample is neutralized, and finally incubated with REPLI-g master mix at 30°C.
Whole genome sequencing of the Bacillus subtilis genome was performed. For analysis, 2 μg of genomic DNA or DNA amplified from 105 cells using the REPLI-g Midi Kit was sheared into 300 bp fragments. For library preparation, 1 μg of each was used. Sequencing was performed on the Illumina MiSeq instrument. [A] Comparable sequence coverage was observed for both gDNA and REPLI-g amplified DNA. [B] Alignment comparison of the genomic loci sequence demonstrates comparably high percentage of alignment for REPLI-g amplified DNA in comparison to the gDNA, which is an indication of minimized levels of junk DNA after WGA (whole genome amplification). Comparison of nonamplified and REPLI-g amplified DNA revealed error rates (mismatch, high-quality error, indels, or chimeras) in a similar percentage range. (Alignment comparison performed using SMALT [Welcome Trust Sanger Institute]).
Twenty DNA samples amplified using REPLI-g technology, without subsequent DNA purification, were subjected to genotyping analysis using 3 STR loci (CSF1PO, TPOX, and THOI). Results were compared with those obtained for unamplified genomic DNA. The DNA was separated by polyacrylamide gel electrophoresis, and visualized by silver staining. A lane with one band represents a homozygote, while a lane with two bands represents a heterozygote for the specific STR locus.
[A] Upon encountering secondary DNA structures, Taq polymerase may pause synthesis, slip, or dissociate from the template. This can result in inaccurate DNA amplification, incomplete loci coverage, and short fragment sizes. [B] REPLI-g Kits utilize Phi29 polymerase, which displaces secondary structures enabling accurate and highly uniform amplification of the entire genome.
Various starting materials, including genomic DNA, and heparin- and EDTA-preserved whole blood, were amplified using REPLI-g Midi and Mini Kits. Typical yields of 40 µg (Midi Kit) and 8–10 µg (Mini Kit) were obtained.