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.

The cloning of polymerase chain reaction (PCR) products provides one with a stable form of the amplified segment and facilitates further manipulation and study of the target molecule. A number of cloning protocols have been developed either based on a restriction endonuclease recognition site built into the primers or by using the template-independent terminal transferase activity (“extendase” ...

Real-time PCR monitors the amount of amplicon as the reaction occurs. Usually, the amount of product is directly related to the fluorescence of a reporter dye. Because it detects the amount of product as the reaction progresses, real-time PCR provides a wide linear dynamic range, demonstrates high sensitivity, and is very quantitative. The initial amount of template DNA is inversely proportiona...

One of the common methods for cloning polymerase chain reaction (PCR) products is overhanging-end cloning (also known as sticky-end or directional cloning). Frequently, it is not possible to use restriction enzyme sites already present in the amplified product, and primers that encode recognition sites of restriction endonucleases in addition to the specific sequence have to be designed. After ...

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 ...

Prediction programs for general PCR (or random PCR) products had been developed [3, 2]. These programs had been experimentally shown to be highly accurate in its prediction [2]. On the other hand, random PCR have been demonstrating that it is very useful as an elementary technology of genome profiling [1], in which random PCR products (a set of DNA fragments extracted from various parts of geno...

Meat adulterations of different species are undetectable and it is common practice globally. In the field of food analysis, species determination is mostly sufficient, but simultaneous detection of several species in a single food product is desirable. The aim of the study was to distinguish between meats of two different species through PCR-RFLP analysis. The meat of two species were used incl...

Recently, we applied the randomamplified polymorphic DNA polymerase chain reaction (RAPD-PCR) (17), also known as arbitrarily primed PCR (AP-PCR) (16), procedure to fingerprint genomes of various varieties and wild relatives of sugarcane (Saccharum spp.) (4). Unlike restriction fragment length polymorphism (RFLP) (2), RAPD-PCR is a simpler, faster, less laborious and less expensive procedure. H...

A restriction site-free cloning method has been developed for inserting a PCR product into a vector flexibly and precisely at any desired location with high efficiency. The method uses a pair of DNA integration primers with two portions. The 3' portion isolates the inserts by PCR, and the 5' portion integrates the PCR products into the homologous region of the vector. For mutagenesis, a third p...

MOTIVATION PCR amplification of highly homologous genes from complex DNA mixtures is known to generate a significant proportion of chimeric sequences. Ribosomal RNA genes are used for microbial species detection and identification in natural environments, and current assessments of microbial diversity are based on these sequences. Thus, chimeric sequences could lead to the discovery of non-exis...