6.5: Polymerase Chain Reaction (PCR)

Review

DNA replication is accomplished by the enzyme DNA polymerase which has the following characteristics:

1. DNA polymerase catalyzes extension of an oligonucleotide in the 5'→3' direction. It cannot go in the opposite direction.
2. A DNA template (i.e. single strand oligonucleotide) is required. This is the information that is replicated (via Watson-Crick base pairing). If you want to replicate the "sense" strand of a DNA duplex, then the template is the anti-sense strand. Since DNA is a duplex, held together by non-covalent forces, we can separate the strands of the duplex (and obtain an appropriate template) by heat (or alkali) denaturation.
3. DNA polymerase can only extend an existing oligonucleotide, it cannot initiate replication. Thus, in addition to a template, DNA polymerase requires a primer oligonucleotide. DNA polymerase extends the primer in the 5'→3' direction.
4. Nucleotides incorporated into the nascent oligonucleotide must be nucleoside triphosphates (i.e. dNTP's). The energy released in the hydrolysis of the phosphate anhydride bonds is used in the formation of covalent linkages (incorporation of the a-phosphate into the phosphodiester backbone of the growing oligonucleotide)
5. Although DNA polymerases catalyze extension in the 5'→3' direction, some have the ability to "proof-read". "Proof reading" is the ability to recognize a misincorporation of a base, and the ability to backup and excise the incorrect base, and then continue on.

Figure 6.5.1: DNA synthesis

Polymerase Chain Reaction (PCR)

• PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence.
• PCR amplification is achieved by using oligonucleotide primers. These are typically short, single stranded oligonucleotideswhich are complementary to the outer regions of known sequence.

Figure 6.5.2: PCR amplification

The oligonucleotides serve as primers for DNA polymerase and each of the denatured strands of the parental DNA duplex serves as the template.

This results in the synthesis of new DNA strands which are complementary to the parent template strands.

The steps of:

1. Template denaturation
2. Primer annealing
3. Primer extension

comprise a single "cycle" in the PCR amplification methodology.

• After each cycle the newly synthesized DNA strands can serve as templates in the next cycle (the PCR primers are typically added in substantial molar excess to the template DNA)

Summary of products at the end of each PCR cycle:

Figure 6.5.3: PCR products

The desired PCR product will be a duplex of the defined length fragment. The question is: how many will be produced?

· The expected amplification of the desired defined length product with respect to the original template concentration 'x' can thus be represented by the formula:

[(2ⁿ - (n + 1)) - (n + 1)] x

or

(2ⁿ - 2(n + 1))

(this is often abbreviated to a simple rule of thumb for the amplification: (2ⁿ - 2n) x)

· The interpretation of this formula is that

• For a given number of cycles 'n' we make '2ⁿ x' total possible duplexes
• For a given number of cycles there will be '2(n+1) (or 2n in our approximation) x' duplexes which are formed from either the original template, or a fragment of indeterminate length, along with a fragment of defined length (and represent an undesired product)
• Thus, the total concentration of desired product (duplexes with a length defined by the PCR primers) will be
  (2ⁿ - 2(n+1)) x (where x is the concentration of the original duplex)

The theoretical amplification value is never achieved in practice. Several factors prevent this from occuring, including:

1. Competition of complementary daughter strands with primers for reannealing (i.e. two daughter strands reannealing results in no amplification).

2. Loss of enzyme activity due to thermal denaturation, especially in the later cycles

3. Even without thermal denaturation, the amount of enzyme becomes limiting due to molar target excess in later cycles (i.e. after 25 - 30 cycles too many primers need extending)

4. Possible second site primer annealing and non-productive priming

Thermal cycling parameters

The thermal cycling parameters are critical to a successful PCR experiment. The important steps in each cycles of PCR include:

1. denaturation of template (typically performed at highest temp - 100°C)

2. annealing of primers (temperature is chosen based upon melting temperature of primer)

3. extension of the primers (performed at optimum for the polymerase being used)

A representative temperature profile for each cycle might look like the following:

Figure 6.5.4: Thermal cycling

Buffers and MgCl₂ in PCR reactions

A typical reaction buffer for PCR would something like:

• 10 mM Tris, pH 8.3
• 50 mM KCl
• 1.5 mM MgCl₂
• 0.01% gelatin
• The MgCl₂ concentration in the final reaction mixture is usually between 0.5 to 5.0 mM, and the optimum concentration is determined empirically (typically between 1.0 - 1.5 mM). Mg²⁺ ions:
  • form a soluble complex with dNTP's which is essential for dNTP incorporation
  • stimulate polymerase activity
  • increase the Tm (melting temperature) of primer/template interaction (i.e. it serves to stabilize the duplex interaction

Generally,

• low Mg²⁺ leads to low yields (or no yield) and
• high Mg²⁺ leads to accumulation of nonspecific products (mispriming).

Choice of Polymerases for PCR

• One of the important advances which allowed development of PCR was the availability of thermostable polymerases.
• This allowed initially added enzyme to survive temperature cycles approaching 100 °C.

• Properties of DNA polymerases used in PCR

	Taq/Amplitaq®	Vent™	Deep Vent™	Pfu	Tth	ULTma™
95 °C half-life	40 min	400 min	1380 min	>120 min	20 min	>50 min
Extension rate (nt/sec)	75	>80	?	60	>33	?
Resulting ends	3' A	>95% blunt	>95% blunt	blunt	3' A	blunt
Stranddisplacement		+	+
5'®3' exo	+				+
3'®5' exo		+	+	+		+

Primers

Primer design

• Generally, primers used are 20 - 30 mer in length. This provides for practical annealing temperatures (of the high temperature regimen where the thermostable polymerase is most active).
• Primers should avoid stretches of polybase sequences (e.g. poly dG) or repeating motifs - these can hybridize with inappropriate register on the template.
• Inverted repeat sequences should be avoided so as to prevent formation of secondary structure in the primer, which would prevent hybridization to template
• Sequences complementary to other primers used in the PCR should be avoid so as to prevent hybridization between primers (particularly important for the 3' end of the primer)
• If possible the 3' end of the primer should be rich in G, C bases to enhance annealing of the end which will be extended
• The distance between primers should be less than 10 Kb in length. Typically, substantial reduction in yield is observed when the primers extend from each other beyond ~3 Kb.

Melting temperature (Tm) of primers

• The Tm of primer hybridization can be calculated using various formulas. The most commonly used formula is:

(1) Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C]

• Examples of T_m calculations

	G	A	T	C	Tm
15 mer	3	5	2	5	46
20 mer	6	5	4	5	62
30 mer	8	6	8	8	92

If the annealing temperature is too high, the primers will not anneal. If it is too low, mispriming can occur at sites of similar DNA sequence to the intended primer binding site

Analysis of the PCR experiment

• 25-30 cycles are common
• Major product should be a duplex DNA molecule whose 5' and 3' ends are defined by the two PCR primers
• Other products can, however, be produced. These will often represent products of secondary priming sites (mispriming) or misincorporation errors of the polymerase
• Thus, PCR product can be heterogeneous and must be separated and analyzed
• Agarose gel electrophoresis, in combination with ethidium bromide staining, is the most common method to separate and analyze PCR products