Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 4

BASIC PRINCIPLES OF THE POLYMERASE CHAIN REACTION (PCR)

101

are easily denatured by the high temperatures needed

to separate the two strands

of

du-

plex DNA. Unlike DNA, however, proteins like DNA polymerases, once denatured, are

generally reluctant to renature. Thus, with each cycle of amplification, it was necessary to

add fresh polymerase for efficient performance. This problem was relieved when DNA

polymerases from thermophilic organisms became available. The most widely

used

of

these enzymes in PCR is from

Thermis acquaticus,

the

Taq

polymerase. This enzyme has

an optimal temperature for polymerization in the range of 70 to 75°C. It extends DNA

chains at a rate of about 2 kb per minute. Most important, it is fairly resistant to the con-

tinual cycles of heating and cooling required for PCR. The half-life for thermal denatura-

tion of

Taq

polymerase is 1.6 h at 95°C, but it is less than 6 minutes at 100°C. When very

high denaturation temperatures are needed,

as in the PCR amplification of very

G

C

rich DNA,

it is sometimes useful to employ

even more thermal-stable polymerases such

as enzymes isolated from organisms that

grow in the superheated water (temperature

above 100°C) near geothermal vents. Such enzymes are called

vent

polymerases.

Examples are the enzyme from

Thermococcus litoralis

with a half-life of 1.8 h at 100°C

and the enzyme from

Pyrococcus furiosis

with a half-life of 8 h at 100°C. However, these

enzymes are not as processive as

Taq

polymerase. This means that they tend to fall off

their template more readily, which makes it more difficult to amplify long templates.

With

Taq

polymerase typical PCR cycle parameters are

DNA denaturation

92–96°C for 30 to 60 seconds

Primer annealing

55–60°C for 30 seconds

Chain extension

72°C for 60 seconds

The number of cycles used depends on the particular situation and sample concentrations,

but typical values when PCR is efficient are 25 to 30 cycles. Thus the whole process takes

about an hour. Typical sample and reagent concentrations used are

Target

10 15 mol, or down to as little as 1 mol

Primer

2

10 11 mol

dpppN’s

2

10 8 mol of each

These concentrations imply that the reaction will eventually saturate once the primer and

dpppN’s are depleted.

There are a few peculiarities of the

Taq

polymerase that must be taken into

considera-

tion in

designing PCR

procedures

or experimental protocols based

on

PCR.

Taq

poly-

merase has no 3

-proofreading exonuclease activity. Thus it can, and does, misincorporate

bases. We will say more

about this

later.

Taq

polymerase does have a 5

-exonuclease ac-

tivity. Thus it will nick translate, which is sometimes undesirable. However, mutants exist

that remove this activity, and they can be used if necessary. The most serious problem

generally

encountered is

that

Taq

polymerase

can

add

an extra nontemplate-coded A

to

the 3 -end of DNA chains, as shown in Figure 4.3

a . It can also, perhaps using a related

activity, make primer dimers, which may or may not contain additional uncoded residues

as shown in Figure 4.3

b. This is a serious problem because once such dimeric primers are

created, they are efficient substrates for further amplification. These primer dimers are a

major source of artifacts in PCR. However, they are usually short and can be removed by

gel filtration or other sizing methods to prevent their interference with subsequent uses of

the PCR reaction product.

NOISE IN PCR: CONTAMINATION

103

nified, especially

when

they

occur early in the reaction. There are many sources of noise

in PCR. One extremely common source is variations in the temperature at different posi-

tions in typical thermal cycling blocks. A typical apparatus for PCR uses arrays of tube

holders (or microtitre plate holders) that can be temperature controlled

by

heating

ele-

ments and a cooling bath, by thermoelectric heating and cooling, by forced convection, or

by switching among several pre-equilibrated water baths. A number of different funda-

mental designs for such thermal cyclers are now available,

and

the

serious practitioner

would be well advised to look carefully at the actual temperature characteristics of the

particular

apparatus used.

For extremely

finicky samples,

such

as

those

that

need

very

G C rich primers, it may be necessary to use the same sample well each time to provide

reproducible PCR. It is for reasons like this that PCR, while it has revolutionized the han-

dling of DNA in research laboratories, has not yet found broad acceptance in clinical di-

agnostic laboratories despite its potential power.

A major source of PCR noise appear to lie with characteristics of the samples them-

selves. The

worst

problem

is undoubtedly sample contamination. If the same sample is

used repetitively in PCR assays, the most likely source of contamination is the PCR reac-

tion product from a previous assay. With PCR we are dealing with a system that amplifies

a DNA sequence by 10

7 fold. Thus, even if there is one part per million in carry over con-

tamination; it will completely dominate the next round of PCR. A second major source of

contamination is DNA from organisms in dust or from DNA shed by the experimenter, in

the form of dander, hair follicles, sweat, or saliva. This problem is obviously of greatest

significance when the samples to be amplified contain human sequences.

The basic cure for most contamination is to carry out PCR under typical biological

containment

procedures

and

use good sterile technique including plugged pipetmen tips,

to prevent contamination by aerosols, and laminar flow hoods, to minimize the ability of

the investigator to inadvertently contaminate the sample. However, these approaches are

not always sufficient to deal with the contamination caused by previous PCR experiments

on similar samples. This situation is extremely frustrating because it is not uncommon for

neophytes to have success with procedures that then progressively deteriorate as they gain

more experience,

since

the overall level

and dispersal of

contaminant in

the

laboratory

degrades DNA at each incorporated dU. Normally these

incorporations are rare muta-

genic events, and the lesion introduced into the DNA is rapidly repaired.

DUG is used in PCR by carrying out the initial PCR amplification using dpppU instead of

dpppT. The amplification product can be characterized in the

normal

way; the properties of

DNA with T fully substituted by dU are not that abnormal except for

a lower melting temper-

PCR is also performed with dpppU, its subsequent carryover can also

be prevented by a

DUG treatment, and this procedure can be repeated

ad libertum.

106

METHODS FOR IN VITRO DNA AMPLIFICATION

MISINCORPORATION

We mentioned earlier that

Taq

polymerase has no 3

editing exonuclease. Because of this

it has a relatively high rate of misincorporation when compared with many other DNA

polymerases. Thus the products of PCR reactions accumulate errors. The misincorpora-

tion

rate

of

Taq

polymerase has

been

estimated

as 1.7

10 4

to 5 10 6 per nucleotide

per cycle. The error rate depends quite a bit on the reaction conditions, especially on the

concentration of dpppN’s used, and on the sequence of the target. The impact of these mi-

spairing rates on the product are straightforward to calculate. At any site in the target, the

fraction of correct bases after n cycles of amplification will be

X corr

(1 X mis

)n 1 nX

mis

where

X mis

is the fraction of misincorporation rate at that site for a single cycle. For 30 cy-

10 3 to

cles

the

fraction of misincorporated

bases

at any

site

will

be

30

X

mis

5.1

1.5

10 4 , using the numbers described above. Thus at any

site the correct sequence is

still overwhelmingly predominant.

However, if one asks, instead, for the amplification of a DNA of length,

L ,

how

many

incorrect bases will each DNA product molecule have after

n

steps, the number is

LnX mis .

With

L 1000,

this means that products

will have

from

0.15 to 5

incorrect

bases. How

serious a problem is this? It depends on the use to which the DNA will be put. As a hy-

bridization probe, these errors are likely to be invisible. If one sequences the DNA di-

rectly, the errors will still be invisible (except for the rare case where a misincorporation

occurred in the first round or two of the amplification and then was perpetuated). This is

because the errors are widely distributed at different sites on different molecules, and se-

quencing sees only the average occupant of each site. However, if the PCR

products

are

cloned, the impact of misincorporation is much more serious. Now, since each clone is the

immortalization of a single DNA molecule, it will contain whatever particular errors that mol-

ecule had. In general, it is a hazardous idea to clone PCR products and then sequence them.

The sequences will almost always have errors. Similarly PCR starting from single DNA mol-

ecules is fine for most analyses, but one cannot recommend it for sequencing because, once

again, the products are likely to show a significant level of misincorporation errors.

LONG PCR

A number of factors may limit the ability of conventional PCR to amplify long DNA tar-

gets. These include depurination of DNAs at the high temperatures used for denaturation,

inhibition of the DNA polymerase by stable intramolecular secondary structure in nomi-

nally single-stranded templates, insufficient time for strand untwisting during convention-

ally used denaturation protocols, as described in Chapter 3, and short templates. The first

problem can be reduced by using increased pH’s to suppress purine protonation, a precur-

sor to depurination. The second problem can be helped somewhat by adding denaturants

like dimethyl sulfoxide (DMSO). The third problem can be alleviated by using longer de-

naturation times. The last problem can be solved by

preparing DNA

in agarose (Chapter

5). However, the most serious obstacle to the successful PCR amplification of long DNA

targets rests in the properties of the most commonly used DNA polymerase, the

Taq

poly-

merase.

SINGLE-SIDED

PCR

107

Taq

polymerase

has

a

significant

rate of misincorporation as described above.

However, it lacks a 3

proofreading exonuclease activity. Once a base is misincorporated

at the 3 -end, the chances that the

extension

will

terminate

at

this

point

become

markedly enhanced. This premature chain termination ultimately leads to totally ineffec-

tive PCR amplification above DNA sizes of 5 to 10 kb. To circumvent the problem of pre-

mature chain termination, Wayne Barnes and coworkers have added trace amounts of a

second thermally stable DNA polymerase like

Pfu,

Vent, or Deep Vent that possesses a

3 -exonuclease activity. This repairs any

terminal mismatches

left

by

Taq

polymerase,

and then the latter can continue chain elongation. With such a two-enzyme procedure,

successful PCR amplification of DNA targets in the 20 kb to 40 kb range are now becom-

ing common.

INCORPORATING

EXTRA FUNCTIONALITIES

PCR offers a simple and convenient way to enhance or embroider the properties of DNA

molecules. As shown in Figure 4.6, primers can be pre-labeled with radioisotopes, biotin,

or fluorescent dyes. Thus the ends of the amplified targets can be derivatized as an intrin-

sic part of the PCR reaction. This is extremely

convenient for many

applications. Since

two primers are chosen, two different labels or tags can be used. One frequent and every

effective strategy is to put a capture tag on one primer—like a biotin—and a detection tag

on the other—like a fluorophore. After the amplification, the product is captured and ana-

lyzed. Only double-stranded material that is the result of amplification that incorporated

both of the primers should be visible.

PCR can also be used to modify the DNA sequence at the ends of the target. For exam-

ple, as shown in Figure

4.6

b,

the

primer can overhang

the ends

of the desired target. As

successive amplification cycles are carried out, the DNA duplexes that accumulate will

contain the target sequence flanked by the additional segments of primer sequence. This

has a number of useful applications. It allows any restriction sites needed to be built into

the primer. Then, as shown in the figure,

after the PCR reaction the product can be

cleaved at these sites for subsequent ligation or cloning steps.

Another use for overhanging primers

arises

in

circumstances

where

the original

amount of known sequence is too short or too imperfect to allow efficient amplification.

This problem arises, for example, when a primer is made to an imperfectly repeating se-

quence. The usual desire in such experiments is to amplify many different copies of the

repeat (e.g., to visualize human DNAs among

a background of rodent DNA in a hybrid

cell as illustrated in Chapter 14), but few of the repeats match the primer well enough to

really give good amplification. By having

an overhanging primer, after the first few

rounds of amplification, the complements to the primer sequence now contain the extra

overhang (Fig.

4.6

c ). The resulting template-primer complexes are much more stable and

amplify much more effectively.

SINGLE-SIDED PCR

A major limitation in conventional PCR is that known DNA sequence is needed on both

sides of the desired target. It is frequently the case, as shown in Figure 4.7

a , that a known

sequence

is

available

only

at

one place

within

the

desired

target,

or

at

one

end of

it.