Convenient single-step, one tube purification of PCR products for direct sequencing

Convenient single-step, one tube purification of PCR products for direct sequencing.

Structural alterations of the apolipoprotein E (apo E) are known to influence the lipid metabolism. The three most common isoproteins Arg 158) are encoded by three co-dominant alleles (e2, el, e4). Phenotyping is cumbersome and, therefore, several methods for apo E genotyping have been developed. Of these, PCR-RFLP (restriction fragment length polymorphism) is currently most rapid and cost-effe...

An efficient method for purification of PCR products for sequencing.

A high-throughput DNA sequencing method that generated high quality data was developed. A frame fashioned from a standard agarose gel combined with 0.1%-0.2% low-melting point (LMP) agarose gel was used to isolate the PCR product of interest. Collected PCR products were centrifuged without any reagents and the supernatants were directly used for a sequencing reaction. This method is simple and ...

Gel Purification of PCR Products. Fatemeh Mehrpooyan*

Direct sequencing of PCR products using unlabeled primers.

An improved protocol is described for using lambda exonuclease to directly sequence PCR products. It is important not to execute PCR cycles beyond the plateau of amplification. The asymmetric PCR and double-stranded DNA sequencing by a snap-cooling procedure were also performed using the same DNA samples and primers. The improved method was the most reliable and produced the best results.

Direct sequencing of PCR products in agarose gel slices.

Direct DNA sequencing of amplified polymerase chain reaction (PCR) products offers several advantages over cloning of amplified DNA products. It is faster (1 day versus 3—5 days) and in DNA samples containing sequence polymorphisms both the normal and mutated sequence can be detected in the same sequencing reaction. The major problems encountered in direct sequencing of amplified DNA have been ...