Thermal cycle labeling: zeptomole detection sensitivity and microgram probe amplification using CviJl* restriction-generated oligonucleotides.
A new method for efficiently labeling and amplifying DNA probes from anonymous samples has been developed. The two/three base recognition endonuclease CviJI* restricts DNA to numerous small fragments primarily 20-60 bp in size. Thermal denaturation of these fragments results in sequence-specific oligonucleotides complementary to their cognate template. Repeated cycles of denaturation, annealing, and extension of such a multiprimed template by a thermostable DNA polymerase results in a significant amplification of the starting material. This method of amplification, referred to as thermal cycle labeling (TCL), appears to generate a large fraction of rearranged and presumably branched products. The inclusion of nucleotide analogs in the TCL reaction generates microgram amounts of haptentagged probe with a detection limit of 25 zmol (2.5 x 10(-20) mol). Reactions containing [alpha-33P]dCTP yield high-specific-activity probes (2.6 x 10(9) cpm/microgram) with reduced radiolytic decay and a useful shelf life of 1 month. CviJI* -generated primers circumvent the need for synthetic oligos while providing microgram amounts of amplified and labeled probes using the described TCL protocol.
Swaminathan
N
N
CHIMERx, Madison, Wisconsin 53704, USA.
McMaster
K
K
Skowron
P M
PM
Mead
D A
DA
D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005821:Genetic Techniques; D007553:Isotope Labeling; D008832:Microchemistry; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000592:standards*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013696:Temperature
D015335:Molecular Probes; D009841:Oligonucleotides; C075913:endodeoxyribonuclease CviJI; D015252:Deoxyribonucleases, Type II Site-Specific