Polymerase chain reaction primer sequences

Abbreviations RFLP, restriction fragment length polymmphism SSP-PCR, polymerase chain reaction using sequence-specific primers SNP, single nucleotide polymorphism. 

Figure 40-7. The polymerase chain reaction is used to amplify specific gene sequences. Double-stranded DNA is heated to separate it into individual strands. These bind two distinct primers that are directed at specific sequences on opposite strands and that define the segment to be amplified. DNA polymerase extends the primers in each direction and synthesizes two strands complementary to the original two. This cycle is repeated several times, giving an amplified product of defined length and sequence. Note that the two primers are present in excess.

Wu, Z., Nagano, I. and Takahashi, Y. (1998) The detection of Trichinella with polymerase chain reaction (PCR) primers constructed using sequences of random amplified polymorphic DNA (RAPD) or sequences of complementary DNA encoding excretory-secretory (E-S) glycoproteins. Parasitology 117, 173-183. 

Oligonucleotide synthesis is used to produce small specific sequences of DNA. These are particularly important as primers in the polymerase chain reaction. 

Figure 13.16 The polymerase chain reaction for the amplification of DNA sequences. DNA is heated to separate the two strands. A primer is attached to the 5 end of each strand and extended using DNA polymerase 1. The two new strands are separated as before and the cycle repeated up to 30 times.

Polymerase chain reaction (PCR) The process by which a specific sequence of DNA can be amplified (copied many times) in vitro. It requires a pair of primers and template DNA, thermostable DNA polymerase (e.g. Taq polymerase), deoxynucleotide triphosphates and a thermocycler. The process can amplify large... 

One of the in vitro (in the test tube) processes used to clone DNA is called the polymerase chain reaction (PCR). A vial in which PCR is to be carried out contains all the necessary components for DNA duplication the piece of DNA to be cloned large quantities of the four nucleotides, A, T, C, G large quantities of a primer sequence, a short sequence of about 20 nucleotides synthesized by the primase enzyme and DNA polymerase.To conduct the process, the vial is hrst heated to 90-95°C for 30 seconds to separate the two DNA chains in... 

Primers are used for direct sequencing following amplification from genomic DNA, unless otherwise indicated. PCR, polymerase chain reaction. 

Fig. 9.4 Establishment of the quantitative methylation-specific polymerase chain reaction (MSP) analytical procedure. The ABI PRISM 7900 HT Sequence Detector system was used to perform real-time polymerase chain reaction (PCR) using MSP primers and bisulfite-modified template DNA. Upper panels Setting up the conditions to obtain the standard curves with 50% (A) or 25% (B) sequential dilution of the template. Lower panels The amplification curves on the left represent P-actin, unmethylated, and methylated MSP products, respectively, for reelin (RELN) (C). Amplification curves were compared at the set threshold before 40 cycles. Amplification curves from various samples are shown in the lower panel right (D)...

In situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ahility to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1-10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13). 

A set of sense and antisense primers should be selected to synthesize specific cDNAs and also to detect the amplified messages of the genes, complementary to their specific gene sequences. It is important to consider the following points while designing the primers for reverse transcriptase and polymerase chain reaction ... 

Figure 3.25 Polymerase chain reaction. The steps involved in the chain reaction are as follows (i) Incubation of the DNA at a temperature above 90 °C in order to separate the two strands of the DNA duplex, (ii) Cooling of the solution to about 50 °C to allow annealing of the primers to the template (i.e. the nucleotides bind to the template DNA according to the basepairing rules), (iii) Finally, addition of the polymerase and Mg ions to extend the nucleotide primer and complete the synthesis of the complementary DNA, which takes place at about 70 °C. (iv) The sequence (i) to (iii) is repeated to allow another extension to occur many repetitions can be carried out which results in enormous multiplication of the DNA strands. NTPs - deoxyri-bonucleoside triphosphates.

Armed with this new tool, Schena et al. (1996) created a microarray of 1,046 human cDNAs of unknown sequence. They were derived from human peripheral blood lymphocyfes fransformed wifh Epsfein-Barr virus. Suitably sized inserts [>600 base pairs (bp)] were cloned into a lambda vector, subsequently infected into an Escherichia coli strain, and finally amplified by polymerase chain reaction (PCR) using 5 -amino-modified primers. The resulting 5 -amino-modified cDNA amplicons were then arrayed onto sily-lated microscope slides. Next, the expression levels in human Jurkat cells undergoing heat shock or phorbol ester induction were examined. 

The polymerase chain reaction (PCR) is an important procedure in genetic engineering that allows any DNA segment to be replicated (amplified) without the need for restriction enzymes, vectors, or host cells (see p. 258). However, the nucleotide sequence of the segment has to be known. Two oligonucleotides (primers) are needed, which each hybridize with one of the strands at each end of the DNA segment to be amplified also needed are sufficient quantities of the four deoxyribonucleo-side triphosphates and a special heat-tolerant DNA polymerase. The primers are produced by chemical synthesis, and the polymerase is obtained from thermostable bacteria. 

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