EXPERIMENT 24

Amplification of a DNA Fragment Using Polymerase Chain Reaction

Theory Polymerase chain reaction (PCR) is a technique that allows the amplification of a specific fragment of double-stranded DNA in a matter of hours. This technique has revolutionized the use of molecular biology in basic research, as well as in a clinical setting. PCR is carried out in a three-step process (Fig. 24-1). First, the template DNA that contains the target DNA to be amplified is heated to denature or “melt” the double-stranded DNA duplex. Second, the solution is cooled in the presence of an excess of two single-stranded oligonucleotides (primers) that are complementary to the DNA sequences flanking the target DNA. Since DNA synthesis always occurs in the 5! to 3! direction (reading the template strand 3! to 5!), you must ensure that the two primers are complementary to (will anneal to) opposite strands of the DNA duplex that flank the region of target DNA that is to be amplified. Third, a heat-stable DNA polymerase is added, along with the four deoxyribonucleotide triphosphates (dNTPs), so that two new DNA strands that are identical to the template DNA strands can be synthesized. If this melting, annealing, and polymerization cycle is repeated, the fragment of double-stranded DNA located between the primer sequences can be amplified over a millionfold in a matter of hours. The heat-stable DNA polymerase ( Taq) commonly used in PCR reactions was isolated from a thermophilic bacterium, Thermus aquaticus. Since this enzyme is heat-stable, it can withstand the high temperatures required to denature the DNA template after each successive round of polymerization

and retain its activity. Since the development of the technique, biotechnology companies have developed a number of improved and specialized polymerase enzymes for use in PCR (Table 24-1). Many of these polymerases are marketed as being more processive and/or “accurate” than the traditional Taq enzyme, since they display 3! to 5! exonuclease (proofreading) activity. The Taq DNA polymerase has no proofreading activity, increasing the possibility of introducing point mutations (single base pair changes) in the amplified DNA product. In this experiment, you will amplify a fragment of pBluescript II (a plasmid), which includes the multiple cloning site (MCS) of the vector (Fig. 24-2). The pBluescript II plasmid comes in the S/K form and the K/S form. These two plasmids are identical except for the orientation of the MCS (see Fig. 24-2). Using restriction enzymes and agarose-gel electrophoresis, you will determine which of these two plasmids was used as a template in the PCR reaction. The sequences of the two primers that will be used in the PCR reaction are shown under “Supplies and Reagents.” Primer 1 will anneal to positions 188 to 211 (5! to 3!) on one strand of the plasmid, while Primer 2 will anneal to positions 1730 to 1707 (5! to 3!) on the opposite strand of the plasmid (Fig. 24-3). On amplification, a 1543-base-pair fragment of DNA will be produced that includes the multiple cloning site of the plasmid. SstI (an isoschizomer of SacI) and KpnI will then be used to determine whether the S/K or K/S form of the pBluescript II plasmid was used as a template in the amplification reaction. Although this experiment is designed to introduce you to the basic technique of PCR, you should be aware that PCR can be used in a variety 385

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SECTION V

Nucleic Acids 5' 3'

3' 5'

Template DNA

Step 1: Denaturation The solution is heated to denature the double-stranded template DNA. 5'

3'

3'

5' Step 2: Primer annealing The solution is cooled in the presence of a high concentration of single-stranded oligonucleotides (primers) that will bind to opposite strands of the template DNA.

5' 3' primer 2 5' 3'

3' 5' primer 1

3' 5' Step 3: Polymerization In the presence of dNTPs, MgCl2, and the appropriate buffer, Taq polymerase will polymerize two new DNA strands that are identical to the template strands.

5'

3'

3' 5' 5'

5'

The cycle is repeated numerous times to achieve exponential amplification of the DNA sequence located between the two primers.

Figure 24-1 The basic principle underlying the technique of polymerase chain reaction (PCR).

Table 24-1 Some Commercially Available Polymerase Enzymes for Use with Polymerase Chain Reaction Polymerase Enzyme

Pfu Taq 2000 Exo" Pfu AmpliTaq UlTma rTth Platinum Taq Vent

Relevant Features

Low error rate (proofreading) Produces blunt-ended PCR products High processivity (for long PCR products) Specially designed for use with PCR sequencing High polymerase temperature (minimizes false-priming) Excellent proofreading activity High processivity (for PCR products 5–40 kb in length) Temperature activation of polymerase (minimizes false priming) Excellent proofreading activity

Company

Stratagene Stratagene Stratagene Perkin Elmer Perkin Elmer Perkin Elmer Life Technologies New England Biolabs

387

1 (1 88 –2 11 )

am pR

Prime r

r2

MC S

Pri m e

lacZ

pBluescript II S/K (2961 base pairs)

KpnI (657)

(17 l

–1

co

30

SacI (759) E1

70

7

)

ori

gin

1 (1 88 –2 11 )

am pR

Prime r

r2

MC S

Pri m e

lacZ

pBluescript II K/S (2961 base pairs)

SacI (657)

(17 l

–1

co

30

KpnI (759) E1

70

7

)

ori

gin

Figure 24-2 Plasmid maps of pBluescriptII (S/K) and pBluescriptII (K/S).

of different applications. One popular application for PCR is its use in the introduction of specific mutations in the product DNA that is amplified from the template DNA. For example, suppose you wanted to produce a PCR product that had restriction-enzyme recognition sequences at either end. If these recognition sequences are present in the single-stranded DNA primers, the target DNA will be amplified to include these sites to allow for

easy cloning into a desired vector following PCR (Fig. 24-4a). In addition to introducing restriction recognition sequences, PCR can be used to add (Fig. 24-4b) or delete (Fig. 24-4c) small sequences from a gene of interest. Provided that the DNA primers are long enough to allow for sufficient base pairing on either side of the desired mutation, nearly any sequence can be added to, or deleted from, a gene of interest. PCR can also be used to

388

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Nucleic Acids

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