Analysis of mutations using PCR and denaturing gradient gel electrophoresis.
Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on primary sequence. Under the appropriate conditions, all base pair (bp) substitutions, frame-shifts, and deletions less than about 10 bp can be resolved from the wild type sequence using DGGE. Polymerase chain reaction (PCR) permits facile amplification of a given region of the genome. We have combined PCR and DGGE to: (i) Localize mutations in the X-linked human androgen receptor gene. PCR/DGGE was used to screen the individual exons in the 2757-bp coding region of the gene in afflicted individuals as well as in potential carriers. Inheritance of a mutant allele has been demonstrated in several cases; (ii) Analyze thousands of thioguanine-resistant mutants simultaneously. The in vitro mutational spectra of MNNG, ICR-191, and cisplatin at the human HPRT locus have been examined by this method. The compounds all have mutational hotspots in a GGGGGG sequence in exon 3; however, the particular mutations induced by the agents were different; (iii) Examine the fidelity of several DNA polymerases used in PCR. The fidelity of Thermus aquaticus DNA polymerase (Taq) is 1-2 x 10(-4) misincorporations/bp/replication. Problems with Taq polymerase arise in the analysis of complex mutant populations by DGGE because the Taq-induced errors reduce the sensitivity of the system. To circumvent this, it had been necessary to use Sequenase, a modified T7 DNA polymerase with a higher fidelity. However, Sequenase is not thermostable and must be added every PCR cycle. A thermostable DNA polymerase from Thermococcus litoralis (Vent) is now available, and we have examined the fidelity of Vent, Taq, and Sequenase polymerase in PCR using DGGE. The fidelity of Vent, Taq, and Sequenase polymerase was 2.4 x 10(-5), 8.9 x 10(-5), and 4.4 x 10(-5) errors/bp, respectively. Vent polymerase had the highest fidelity of the three enzymes tested.
Cariello
N F
NF
Pathology Department, University of North Carolina, Chapel Hill 27599.
Swenberg
J A
JA
De Bellis
A
A
Skopek
T R
TR
D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D002945:Cisplatin / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005796:Genes; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011944:Receptors, Androgen / Q000235:genetics; D013698:Templates, Genetic
D011944:Receptors, Androgen; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D002945:Cisplatin