Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing ha...

PCR is a quick and effective way of identifying the presence and 'affiliation' of bacteriophages, or phage-encoded genes from environmental samples, bacterial cells or purified viruses. The limitations are that you have to know what you are looking for in order to find it. Although the bacteriophage world does not have the advantage of a conserved gene, present in all members, there are many ph...

OBJECTIVE To analyze the sequence difference between human A, B, and O alleles and establish the method of ABO genotyping by PCR direct sequencing. METHODS PCR-direct sequencing technique was used to analyze two regions of cDNA from A transferase gene, 233-433 and 660-788. RESULTS Two nucleotide substitutions at 258th and 297th were found in 233-433 region, and a nucleotide substitution at ...

A method is described to simultaneously amplify and sequence DNA using a new class of nucleotides containing boron. During the polymerase chain reaction, boron-modified nucleotides, i.e. 2'-deoxynucleoside 5'-alpha-[P-borano]-triphosphates, are incorporated into the product DNA. The boranophosphate linkages are resistant to nucleases and thus the positions of the boranophosphates can be reveale...

BACKGROUND Mutations in calreticulin (CALR) have been reported to be key markers in the molecular diagnosis of myeloid proliferative neoplasms. In most previous reports, CALR mutations were analyzed by using Sanger sequencing. Here, we report a new, rapid, and convenient system for screening CALR mutations without sequencing. METHODS Eighty-three bone marrow samples were obtained from 81 pati...

We demonstrate that routine PCR product analytical agarose gels can also serve as preparative gels for quick DNA template purification before sequencing. The band of interest is excised, placed into a Gel Nebulizer inside a Micropure separator and rapidly purified in a single centrifugation step. Gel-purified PCR product, suitable for manual and automated sequencing, is delivered within 10 min.

Direct sequencing of PCR products by T7 DNA polymerase is prone to problems in GC-rich regions, due to the stability of secondary structure and rapid reannealing of the heat-separated complementary strands of DNA. To overcome these difficulties, we examined the effect of adding formamide to the sequencing reaction. Formamide weakens the hydrogen bonding between nucleotides (1, 2), and is, there...

Laboratoire de GCnCtique Microbienne, lnstitut National de la Recherche Ag ro no m iq ue, Do rn a in e de Vilvert, 78352 Jouy en Josas cedex, France Within the Bacillus subtilis genome sequencing project, the region between /ysA and i/vA was assigned to our laboratory. In this report we present the sequence of the last 36 kb of this region, between the kdg operon and the attachment site of the ...

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recen...