Evaluation of a whole-genome amplification method based on adaptor-ligation PCR of randomly sheared genomic DNA.
High-throughput genetic studies often require large quantities of DNA for a variety of analyses. Developing and assessing a whole-genome amplification method is thus important, especially with the current desire for large-scale genotyping in previously collected samples for which limited DNA is available. The method we have developed, called PRSG, is based on an adaptor-ligation-mediated PCR of randomly sheared genomic DNA. An unbiased representation was evaluated by performing PCR on 2,607 exons of 367 genes, which are randomly distributed throughout the genome, on PRSG products of hundreds of individuals. An infrequent loss (<1%) of the exon sequence on the PRSG products was found. Out of 307 microsatellites on various chromosomes, 258 (84%) were amplified in both the PRSG product and an original DNA, whereas 49 (16%) microsatellites were lost only in the PRSG product. Array CGH analysis of 287 loci for measuring the relative gene copy number demonstrated that a low bias was detected. Moreover, this method was validated on 100-1,000 laser-captured cells from paraffin-embedded tissues. These data show that PRSG can provide a sufficient amount of genomic sequence for a variety of genetic analyses as well as for long-term storage for future work.
Tanabe
Chikako
C
Genetics Division, National Cancer Center Research Institute, Tokyo, Japan.
Aoyagi
Kazuhiko
K
Sakiyama
Tokuki
T
Kohno
Takashi
T
Yanagitani
Noriko
N
Akimoto
Shingo
S
Sakamoto
Michiie
M
Sakamoto
Hiromi
H
Yokota
Jun
J
Ohki
Misao
M
Terada
Masaaki
M
Yoshida
Teruhiko
T
Sasaki
Hiroki
H
D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics; D004273:DNA, Neoplasm / Q000737:chemistry* / Q000235:genetics; D015245:Deoxyribonuclease BamHI / Q000235:genetics; D015246:Deoxyribonuclease EcoRI / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D004938:Esophageal Neoplasms / Q000737:chemistry / Q000235:genetics; D018628:Gene Dosage; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D017403:In Situ Hybridization; D018895:Microsatellite Repeats / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic / Q000235:genetics; D016610:Tissue Embedding
D004273:DNA, Neoplasm; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases; D011088:DNA Ligases