A PCR-based amplification method retaining the quantitative difference between two complex genomes.
With the increasing emergence of genome-wide analysis technologies (including comparative genomic hybridization (CGH), expression profiling on microarrays, differential display (DD), subtractive hybridization, and representational difference analysis (RDA)), there is frequently a need to amplify entire genomes or cDNAs by PCR to obtain enough material for comparisons among target and control samples. A major problem with PCR is that amplification occurs in a nonlinear manner and reproducibility is influenced by stray impurities. As a result, when two complex DNA populations are amplified separately, the quantitative relationship between two genes after amplification is generally not the same as their relation before amplification. Here we describe balanced PCR, a procedure that faithfully retains the difference among corresponding amplified genes by using a simple principle. Two distinct genomic DNA samples are tagged with oligonucleotides containing both a common and a unique DNA sequence. The genomic DNA samples are pooled and amplified in a single PCR tube using the common DNA tag. By mixing the two genomes, PCR loses the ability to discriminate among the different alleles and the influence of impurities is eliminated. The PCR-amplified pooled samples can be separated using the DNA tag unique to each individual genomic DNA sample. The principle of this method has been validated with synthetic DNA, genomic DNA, and cDNA applied on microarrays. By removing the bias of PCR, this method allows a balanced amplification of allelic fragments from two complex DNAs even after three sequential rounds of PCR. This balanced PCR approach should allow genetic analysis in minute laser-microdissected tissues, paraffin-embedded archived material, or single cells.
Makrigiorgos
G Mike
GM
Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA. mmakrigiorgos@partners.org
Chakrabarti
Subrata
S
Zhang
Yuzhi
Y
Kaur
Manjit
M
Price
Brendan D
BD
D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics*; D020224:Expressed Sequence Tags; D015894:Genome, Human; D006801:Humans; D008168:Lung / Q000737:chemistry; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011467:Prostate / Q000737:chemistry; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D018076:DNA, Complementary