Reaction polymerase chain

With the polymerase chain reaction (PCR), a few molecules of any DNA sequence can be multiplied by a factor of 10 to 10 in a very short time. The sequence is multiplied in a highly specific way until it becomes visible electrophoretically. Based on PCR, analytical techniques have been developed for species identification in the case of animal and plant foods and microorganisms. It is 

Rape seed Higher concentrations of 12 0 und 14 0, herbicide resistance 

Soybean Altered fatty acid spectrum (cf. 14.3.2.2.5), herbicide tolerance 

Tomato Delayed ripening, increased pectin content 

As the name suggests, polymerase chain reaction (PCR) is a method of amplifying selected segments of target nucleic acid sequences (typically 150-3000 base pairs in length) by several orders of magnitude prior to separation and characterization. Since the pioneering research and 

Because each newly synthesized portion can itself be replicated, target DNA doubles with each replication cycle. Upon completion, the amplified nucleic acid is separated using agarose gel or polyacrylamide gel electro- 

For both scenarios the polymerase chain reaction (PCR) can be used. Alternatively, in the second case, a hybridization technique such as Southern blot (see later) can be applied. 

PCR utilizes the enzymatic properties of a DNA polymerase to duplicate DNA through extension of a short, existing, double-stranded piece of DNA, usually 15-30 bp. This short, double-stranded DNA piece can be mimicked through addition of short oligonucleotides (primer) to each of the DNA strands, the sense (5 —3 ) and anti-sense (3 -5 ) primer. First, in the denaturing step, the DNA has to be 

Mixture of nucleoside triphosphate monomers FIGURE 11.48 The polymerase chain reaction. 

The polymerase chain reaction (PCR), invented in 1986 by Mullis [71], will produce multiple copies of a given DNA sequence. Essential for this process are special polymerase enzymes, of which at least three different varieties are now known. 

More recently, whole genome amplification (WGA), multiple displacement amplification (MDA) and other advanced copying techniques have been evolved [72]. 

In 1935, H. Spemann predicted that it should be possible for an organism to be produced by transmitting the nucleus (i.e. the DNA) of a cell from one adult human to an egg produced by another. 

A few basic components are needed to perform a PCR. First, two specific ofigonucle-otides (15-25 nt), called primers, are needed. They are derived from both strands of the target sequence, thus they determine the size and specificity of the resulting PCR product. The other components are a thermo-stable DNA polymerase, deoxyribonu-cleotides and a defined reaction buffer, which contains magnesium ions as cofactor and estimates the optimal reaction conditions for the polymerization of DNA (Saiki et al., 1988). 

PCR is a cycle reaction and is carried out in a thermocycler. Each cycle consists of three steps. In the first step, denaturation, the initial DNA and primers are healed at 95 C and denatured into single strands. In the second step, called annealing, the primers hybridize to their opposite sequence at both DNA strands. The annealing temperature is usually 3-5 °C below the melting temperature of the primer. If the selected annealing tanperatnre chosen is too low the primers may also bind at positions which are not 100% complanentary and thus lead to nonspecific products. If the temperature is too high the primers do not bind or bind incompletely and no product, or only a small amount, is formed. 

Based on the PCR product detection, PCR can be categorized into endpoint PCR and time-point, called real-time PCR. In endpoint PCR the ready PCR product is visualized by agarose or polyacrylamide gel electrophoresis followed by staining with fluorescent dyes, for instance elhidium bromide or SYBR Green I. 

Target organism Target gene Product Technique Application PrePCR processing Assay sensitivity and time Comments References 

Beer-spoilage lactobacilli horA 342 EP-PCR Pure culture Enzymatic lysis, CTAB and chlo-form extractions, acetate and alcohol precipitations IxlO CFU/mL Good correlation with beer-spoilage ability Sami, Yamashita, Kadokura, Yoda, and Yamasaki (1997) 

PCR amplifies DNA sequences that lie between specific 5 and 3 sequences. 

SITE-DIRECTED MUTAGENESIS can be used to change one or more base pairs in the DNA resulting in a change in the amino acid that appears in the protein produced from this DNA. 

By first making a DNA copy of an RNA molecule, one can also amplify RNA sequences. Because reverse transcriptase (copies RNA to DNA) is used in the first step, this is called RT-PCR. 

Applications of PCR PCR has made a huge impact in molecular biology, with many applications in areas such as cloning, sequencing, the creation of specific mutations, medical diagnosis and forensic medicine. 

Related topics DNA structure (FI) Nucleic acid hybridization (12) DNA replication in bacteria (F3) DNA cloning (13) Restriction enzymes (11) DNA sequencing (15) 

Instant Notes in Biochemistry 2nd Edition, B.D. Hames N.M. Hooper, (c) 2000 BIOS Scientific Publishers Ltd, Oxford. 

The three steps of the PCR cycle are repeated. Thus in the second cycle, the four strands denature, bind primers and are extended. No other reactants need to be added. The three steps are repeated once more for a third cycle (Fig. 1) and so on for a set number of additional cycles. By the third cycle, some of the PCR products (indicated by asterisks in Fig. 1) represent DNA sequence only between the two primer sites and the sequence does not extend beyond these sites. As more and more reaction cycles are carried out, this type of double-stranded DNA molecule becomes the majority species present. After 20 cycles, the original DNA has been amplified a million-fold and this rises to a billion- 

Applications PCR already has very widespread applications, and new uses are being devised 

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CR Polymerase Chain Reaction. Widely used method for amplifying a DNA base sequence... 

Polyethylene (Section 6 21) A polymer of ethylene Polymer (Section 6 21) Large molecule formed by the repeti tive combination of many smaller molecules (monomers) Polymerase chain reaction (Section 28 16) A laboratory method for making multiple copies of DNA Polymerization (Section 6 21) Process by which a polymer is prepared The principal processes include free radical cationic coordination and condensation polymerization Polypeptide (Section 27 1) A polymer made up of many (more than eight to ten) amino acid residues Polypropylene (Section 6 21) A polymer of propene Polysaccharide (Sections 25 1 and 25 15) A carbohydrate that yields many monosacchande units on hydrolysis Potential energy (Section 2 18) The energy a system has ex elusive of Its kinetic energy... 

The plaque assay is desirable because it is very sensitive and only detects infectious viral particles. However, there are viral agents which cannot be supported by cell lines. In these cases other methods must be used. The polymerase chain reaction (PGR), which amplifies DNA or RNA from viral agents, can be used to detect the presence and quantity of viral agents. The amount of RNA or DNA target in the initial sample can be determined by competitive PGR where the quantity of amplified product is compared to a control PGR product where the initial amount of target is known. Quantification is also possible by an end-point dilution method similar to that used to determine a tissue culture infections dose. PGR methods can be very sensitive however. 

Fig. 6. Polymerase chain reaction (PCR) mediated site-directed mutagenesis. The 5 and 3 ends of the nucleotide strands are indicated. The four arrows surrounding the DNA template represent oligonucleotide primers 1—4. See text for discussion.

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

FIGURE 28.14 The polymerase chain reaction (PCR). Three cycles are shown the target region appears after the third cycle. Additional cycles lead to amplification of the target region. 

Polymerase chain reaction (Section 28.16) A laboratory method for making multiple copies of DNA. 

FIGURE 13.21 Polymerase chain reaction (PCR). Oligonucleotides complementary to a given DNA sequence prime the synthesis of only that sequence. Heat-stable Taq DNA polymerase survives many cycles of heating. Theoretically, the amount of the specific primed sequence is doubled in each cycle. 

K. B. Mullis (La Jolla, California) invention of the polymerase chain reaction. 

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