Allelic discrimination by nick-translation PCR with fluorogenic probes.

Nick-translation PCR was performed with fluorogenic probes. Two probes were used: one complementary to a sequence containing the F508 codon of the normal human cystic fibrosis (CF) gene (wt DNA) and one complementary to a sequence containing the delta F508 three base pair deletion (mut DNA). Each probe contained a unique and spectrally resolvable fluorescent indicator dye at the 5' end and a common quencher dye attached to the seventh nucleotide from the 5' end. The F508/delta F508 site was located between the indicator and quencher. The probes were added at the start of a PCR containing mut DNA, wt DNA or heterozygous DNA and were degraded during thermal cycling. Although both probes were degraded, each probe generated fluorescence from its indicator dye only when the sequence between the indicator and quencher dyes was perfectly complementary to target. The identify of the target DNA could be determined from the post-PCR fluorescence emission spectrum.

DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments.

The incorporation of fluorescently labeled dideoxynucleotides by T7 DNA polymerase is optimized by the use of Mn2+, fluorescein analogs and four 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates) (dNTP alpha S's). The one-tube extension protocol was tested on single-stranded templates, as well as PCR fragments which were made single-stranded by digestion with T7 gene 6 exonuclease. Dye primer sequencing using four dNTP alpha S's was shown to give uniform termination patterns which were comparable to four dNTPs. Efficiency of the polymerase also appeared to improve with the dNTP alpha S's. A mathematical model was developed to predict the pattern of termination based on enzyme activity and ratios of ddNTP/dNTPs. This method can be used to optimize sequencing reactions and to estimate enzyme discrimination constants of chain terminators.

Imaging as a tool for improving length and accuracy of sequence analysis in automated fluorescence-based DNA sequencing.

A new method of signal analysis for automated fluorescence-based DNA sequencing is presented. Signal resolution is a limiting factor in obtaining accurate sequence information beyond 400-450 nucleotides per gel lane. We have developed a computer program for the imaging of DNA bands in sequencing gels. The image analysis shows that distortions in the shapes of the bands decrease resolution of peaks observed served in the standard data plots. Reconstruction of the undistorted band shape prior to signal analysis substantially improves the resolution of peaks and may improve the accuracy and length of the contiguous sequence read. Image analysis identified other factors limiting the accuracy and length of automated DNA sequence analysis and provided a tool for evaluating various remedies. Our techniques should also be applicable in other systems, for example, in gel electrophoresis of proteins and DNA restriction fragments, and in scranning densitometry.

Fluorescence detection in automated DNA sequence analysis.

We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific for the bases A, C, G and T. The reaction mixtures are combined and co-electrophoresed down a single polyacrylamide gel tube, the separated fluorescent bands of DNA are detected near the bottom of the tube, and the sequence information is acquired directly by computer.