Restriction endonuclease digestion eliminates product contamination in reverse transcribed polymerase chain reaction.
Restriction endonuclease digestion was used to eliminate false-positive signals caused by polymerase chain reaction (PCR) product DNA contamination in a reverse transcribed (RT) PCR for amplifying rubella virus (RV) RNA sequences. A restriction enzyme selected to cut the PCR product DNA between, but not within, the primer binding sites was used to digest reaction mixtures after reverse transcription but before PCR amplification. Because restriction enzymes generally react only with specific double-strand sequences, contaminating DNA was rendered inactive while reverse-transcribed single strand cDNA was amplified. Assays showed that restriction enzyme digestion reduced template activity of product DNA by a factor of 10(7), while leaving sensitivity of the RT-PCR unaffected.
Dougherty
R M
RM
Department of Microbiology and Immunology, SUNY Health Science Center, Syracuse 13210.
Phillips
P E
PE
Gibson
S
S
Young
L
L
D000818:Animals; D004279:DNA, Viral / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D005189:False Positive Reactions; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012412:Rubella virus / Q000235:genetics / Q000302:isolation & purification*; D014709:Vero Cells
D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific