The advance of Taq-based polymerase chain reaction (PCR) technology (1–3) has had a tremendously positive impact on biomedical research. The combination of PCR and sequencing further revolutionized biological research (3, 4). Sequence amplification technology has provided a spe ...
The acceptance of polymerase chain reaction (PCR) as a routine method in molecular biology has created a need for simple and robust approaches to DNA sequencing of the amplification products. In addition to its ease, direct sequencing of PCR products has the advantage that nucleotide misinc ...
The polymerase chain reaction (PCR) is well known for being a rapid and versatile method for the amplification of defined target DNA sequences. This technique can be applied to a variety of research areas, such as the identification and typing of single nucleotide substitutions of DNA sequence ...
The use of the polymerase chain reaction (PCR) allows isolation and examination of genomic DNA sequences using specific oligonucleotide primers to amplify a target region with Taq DNA polymerase (1,2). The PCR products can be screened for mutations using molecular techniques, such as all ...
The polymerase chain reaction (PCR) facilitates the rapid in vitro amplification of target DNA segments. Numerous applications have been developed to exploit the vast potential of PCR, and many of these require sequence analysis of the DNA product. This chapter describes the dideoxy chai ...
Although numerous methods are now available for direct sequencing of PCR products, cloning of amplified DNA for sequencing in M13 vectors remains an attractive approach because of the high quality of sequence information generated from single-stranded bacteriophage DNA templat ...
Genomic amplification with transcript sequencing (GAWTS) (1,2) is a generally applicable method for direct sequencing of PCR material. GAWTS is centered around the attachment of a phage promoter sequence (T7, Sp6, or T3) to the 5′-end of one or both PCR primers. The phage promoter sequence allows t ...
Because only between 1 and 10% of bacteria present in soil and aquatic environments are culturable using currently available methods, attempts to identify and quantify the nonculturable, majority population must circumvent the need for culture. Molecular biological techniques, ...
The advent of direct sequencing of polymerase chain reaction (PCR) products has permitted extremely rapid analysis of DNA mutants and cDNA clones. However, direct PCR sequencing has been problematic for a number of technical reasons, including the presence of impurities and excess olig ...
The eukaryotic cell cycle is a process in which cells grow and then divide into two genetically identical cells. The cell cycle is divided into four discrete phases allowing for the orderly transition from deoxyribonucleic acid (DNA) replication to chromosomal condensation, spindle fo ...
Haploid yeast cells mate to form a zygote, whose progeny are diploid cells. A fundamentally sexual event, related to fertilization, yeast mating nevertheless exhibits cytological properties that appear similar to somatic cell fusion. A large collection of mutations that lead to defec ...
Hyphal fusion occurs at different stages in the vegetative and sexual life cycle of filamentous fungi. Similar to cell fusion in other organisms, the process of hyphal fusion requires cell recognition, adhesion, and membrane merger. Analysis of the hyphal fusion process in the model organi ...
Differentiation of vegetative cells of the haploid eukaryote Chlamydomonas is dependent on environmental conditions. Upon depletion of nitrogen and exposure to light, vegetative cells undergo a mitotic division, generating gametes that are either mating-type plus (mt) or mati ...
In the nematode Caenorhabditis elegans, 300 of the 959 somatic nuclei present in the adult hermaphrodite are located in syncytia. These syncytia are formed by the fusion of mononucle-ate cells throughout embryonic and postembryonic development. These cell fusions occur in a well-char ...
Myogenic differentiation in Drosophila melanogaster, as in many other organisms, involves the generation of multinucleate muscle fibers through the fusion of myoblasts. Prior to fusion, the myoblasts become specified as one of two distinct cell types. They then become competent to fu ...
Direct sequencing of PCR products (1) has proven to be a powerful method in the generation of nucleic acid sequence data. Using these techniques, it is possible to produce microgram quantities of pure target DNA and subsequently its nucleotide sequence in a few hours, even, theoretically, from one ...
Following the isolation of a clonal recombinant phage or plasmid after screening a cDNA library, the first analyses that are routinely performed are the determination of the insert size and the sequence of the 3′- and 5′-ends of the cloned fragment. Prior to the use of PCR, this was a laborious process, esp ...
The following protocol describes a method that can be used for the direct sequencing of polymerase chain reaction (PCR) products using nonradioactive detection procedures. It is based on our experience with a method we have previously published (1). Direct sequencing of PCR products is gen ...
The use of magnetic particles in many fields of biochemistry, molecular biology, and medicine has been well documented, and several magnetic particles are now available for diagnostic and cell-separation purposes. The solid-phase approaches have improved robustness by their incr ...
The time and labor required for generating nucleotide sequence information has been significantly reduced since the development of the polymerase chain reaction (PCR). PCR allows the production of sufficient amounts of DNA template in vitro, and consequently, the time-consuming c ...
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