Parallel DNA polymerase chain reaction: Synthesis of …

If a person were to attempt extending a synthetic oligonucleotide prepared to be complementary to a target on human DNA by just one base, using DNA polymerase and dideoxynucleoside triphosphates, using four different tubes each containing all four bases, but only one of them in each tube alpha-labeled with 32P, optimistically one might be able to discover the identity of the nucleotide on the DNA target just three-prime of the oligomer. Dideoxy-sequencing worked that way…but…Huge but…that only worked on cloned DNA where the ratio of target to non-target DNA was increased by a factor of about a million. Fortunately for me I was thinking about other things that might go wrong than just the brute improbability that only the right sequence would be engaged. I paid just enough attention to this hypothetical problem to plan on using two oligonucleotides, one designed for each strand of the target sequence coming at the base pair in question from either side. Although these two sides would be far distant in the denatured reaction mixture they would still represent complementary strands and if one told me that a 'T' was three-prime to one oligo, the other should have told me 'A' was three-prime to the other. Not much of a control, but I had oligos to burn. In fact that was what I was trying to do. We had excess oligos on our hands.

of polymerase chain reactions using ..

Chapter 8: In vitro Amplification of DNA by the Polymerase Chain ReactionJump up

Polymerase chain reaction - Wikipedia

Dr. Mullis received a Nobel Prize in chemistry in 1993, for his invention of the polymerase chain reaction (PCR). The process, which Dr. Mullis conceptualized in 1983, is hailed as one of the monumental scientific techniques of the twentieth century. A method of amplifying DNA, PCR multiplies a single, microscopic strand of the genetic material billions of times within hours. The process has multiple applications in medicine, genetics, biotechnology, and forensics. PCR, because of its ability to extract DNA from fossils, is in reality the basis of a new scientific discipline, paleobiology.

Reverse transcription polymerase chain reaction - Wikipedia

DNA strands containing an unnatural T-triazole-T linkage have been synthesized by click DNA ligation between oligonucleotides with 3′-AZT and 5′-propargylamido dT and amplified efficiently by polymerase chain reaction (PCR) using several different polymerases. DNA sequencing of PCR amplicons and clones in two different sequence contexts revealed the presence of a single thymidine at the ligation site. The remarkable ability of thermostable polymerases to reproducibly copy DNA templates containing such an unnatural backbone opens up intriguing possibilities in gene synthesis, genetic analysis, biology, and nanotechnology.

Biography - Dr. Kary Banks Mullis

In the standard PCR technique (Fig. 1), two oligos are designed to be complementary to the end sequences of the DNA to be amplified (the target sequence, or amplicon). These oligos, or PCR "primers," are complementary to opposite strands of the double-stranded DNA and are generally 15 to 25 nucleotides in length. DNA synthesis primed from the two oligos results in selective amplification of the complementary strands of DNA contained between the two primers. Amplification occurs during repetitive cycles of synthesis that repeat the following three steps: (1) denaturation of the double-stranded DNA template; (2) annealing of the two primers to the single-stranded template; and (3) extension of the primers by a thermostable DNA polymerase. In theory, every cycle doubles the number of target sequences, although in practice PCR is not quite that efficient. The reaction mix includes the following: the DNA sample, two primers that "select" DNA to be amplified; the thermostable DNA polymerase to extend the primers; an excess of the four deoxynucleotides A, T, G, and C needed for DNA synthesis; and generally 10 m M Tris-HCl buffer, 50 mM KCl to provide the proper ionic strength, and Mg as a cofactor (optimal Mg concentrations may need to be determined for each reaction). Each step in the cycle is performed at a different temperature, and PCR machines are available to perform the temperature cycling automatically. Denaturation occurs at 94 to 96°C, and extension by polymerase is generally at 72°C. The temperature of the second step, primer annealing, is crucial and depends on the sequence of the primers, the concentration of the components of the PCR mix and the salt concentration. If the temperature is too high, not enough of the primers anneal to template DNA to have an efficient PCR, but a low annealing temperature allows nonspecific priming and results in spurious amplification products. The Tm (temperature when half the molecules are double-stranded and half are single- stranded) is often used for the annealing temperature and then adjusted up or down if necessary. If the primer is 18 bases long or less, the T m can be estimated by the following formula:

Tib Molbiol - Oligonucleotides, Design for PCR and …

Figure 1. Basic PCR. The polymerase chain reaction uses multiple rounds of DNA denaturation, primer annealing, and primer extension to amplify a specific target sequence. First, double-stranded template DNA is denatured by brief incubation at 95°C. Secondly, primers that select the target sequence are annealed to single-stranded template DNA at a temperature determined by the length and sequence of the primers. Finally, the temperatre is raised to 72°C to permit primer extension by a thermostable DNA polymerase, thus producing double-stranded target sequence. Each cycle of PCR repeats the same three steps, using the newly synthesized target DNA as additional PCR template. Thus, each round of PCR theoretically doubles the previous number of target sequences. Millions of copies of the target sequence are synthesized after multiple rounds of PCR. Horizontal arrows represent PCR primers. Double-stranded DNA is represented by horizontal lines joined by short vertical lines. The vertical lines represent the Watson-Crick base pairs.

Clontech Laboratories, Inc. - Life Science Tools and …

There are times when you don't want the temperature changes to be rapid, and here's an example: Suppose we are trying to work out the conditions for a polymerase chain reaction using two oligonucleotides that are approximately 1000-fold degenerate. We would like to use that handy web site to determine the Tm, so we would know what annealing temperature to program into the machine, but we don't actually know which of the 1000 versions of each oligonucleotide will be an exact match to the target sequence. What we would really like to do is to introduce some flexibility into the temperature cycle, so that every potential oligonucleotide has a fair chance of annealing to the target.