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

Figure 4.6

Introducing extra

functionalities by appropriately designed primers.

(a) Incorporation

of a 5 -terminal label (asterisk).

(b)Incorporation of flanking restriction sites, useful for subsequent

cloning.

(c) Compensating for less than optimal length initial sequence information.

This problem typically arises when one is trying to walk by from a known region of the

genome into flanking unknown regions. Then one starts with a bit of known sequence at

the extreme edge of the charted region, and now the goal is to make a stab into the un-

known. There is still not a consensus on the best way to do this, but there have been a few

successes, and many failures.

Consider the simple scheme shown in Figure 4.7

b. The known sequence is some-

where within a restriction fragment of a length suitable for PCR. One can ligate arbi-

trary bits of known DNA sequence,

called

splints

or

adapters,

onto the ends of

the

DNA

fragment of interest. Now, in principle,

the use of

the one known primer and

one

of the two ligated splint primers

will allow selective amplification of

one side

of

the target. This amplification process will work as expected. However, the complica-

tion

arises not from the target but

from

all

the other

molecules in the sample. They

too

are substrates for amplification

using

two

copies of

the splint primer

(Fig. 4.7

c ).

SINGLE-SIDED PCR

109

Figure 4.7

Single-sided PCR.

(a) General situation that requires this method; dashed

line repre-

sents the only known sequence in the target.

(b)A potentially simple scheme.

(c) Unwanted ampli-

fication products that defeat the scheme in

(b).

performed using

only the sequence-specific primer with a capture

tag, like

biotin.

Then, before any additional rounds of amplification are executed, the

product

of

the

first cycle is

physically purified using streptavidin-coated magnetic

beads

or

some

other strepatvidin-coated surface (Fig.

4.8). Now the splint primer is added, along with

nonbiotinylated specific primer, and the

PCR amplification is allowed to proceed nor-

mally. When

the procedure is successful, very pure desired product is achieved, since

all of the

potential side product precursors are removed before they are able to be

amplified.

combination designed so that

the primer will work only after the splint has been

repli-

cated once, and the splint can only be replicated by synthesis initiated at the known bit of

sequence. Two versions of this are shown in Figure 4.9. In one case a splint is used that is

dephosphorylated, so it cannot ligate to itself, and the double-stranded region is short and

A T rich (Fig. 4.9

a ). After this is ligated to the target, the sample

is heated to melt off

the short splint strand. Next one cycle of DNA synthesis is allowed, using only the primer

in the known target sequence. This copies the entire splint and produces a template for the

second splint-specific primer. Both primers are now added, and ordinary PCR is allowed

to proceed. However, only those molecules replicated during the first PCR cycle will be

substrates for subsequent cycles. In the second version, called

bubble PCR, the splint con-

tains a noncomplementary segment of DNA which forms an interior loop (Fig. 4.9

b). As

before, the first round of PCR uses only the sample-specific primer. This copies the mis-

paired template strand faithfully so that when complementary primer is added to it,

nor-

mal PCR can ensue. These procedures are reported to work well. It is worth noting that

they can easily be enhanced by

attaching a capture tag to the target-specific primer in the

REDUCING COMPLEXITY WITH PCR

113

Chapter 3. It is the total amount of different DNA sequence. For single-copy DNA the

complexity determines the rate of hybridization. Thus it can be very useful to selectively

reduce the complexity of a sample, since this speeds

up subsequent attempts to analyze

that sample by hybridization. The problem is that in general, one rarely has enough infor-

mation

about the

DNA

sequences in a sample to choose a large but specific subset

of it

for PCR amplification.

A powerful approach has been developed to use PCR to selectively reduce sample

complexity without any prior sequence knowledge

at all. This approach promises to

greatly facilitate genetic and physical mapping of new, uncharted genomes. It is based on

the use of short, random (arbitrary) primers. Consider the use of a

single

oligonucleotide

primer

of length

n . As shown in Figure 4.11, this primer can produce DNA amplification

only if its complementary sequence exists as an inverted repeat, spaced within a distance

range amenable to efficient PCR. The inverted repeat requires that we specify a DNA se-

quence of 2

n

bases. For a statistically random

genome of

N base

pairs,

the probability of

this occurring at any particular place is

N 4 2n , which is quite small for almost any

n

large

enough to serve as an effective primer. However, any

placement close enough for PCR

will yield amplification products of the two primer sites. If

L

is the

maximum

practical

PCR length, the probability that some observable PCR product will be seen is

LN 4 2n . It

is instructive to evaluate this expression for a mammalian genome with

N 3 109 bp.

For

L 2000 the results are

OLIGONUCLEOTIDE

NUMBER OF

TOTAL AMOUNT OF

LENGTH

PCR PRODUCTS

AMPLIFIED DNA

8

1500

1.5

106 bp

9

100

1.0 105

bp

10

6

6.0 103 bp

These results make it clear that by using single arbitrary short primers, we can

sample

useful discrete subsets of a genome. Each different choice of primer will presumably give

a largely nonoverlapping subset. The complexity of the reaction products can be

con-

trolled by the primer length to give simple or complex sets of DNA probes. This method

has been used, quite successfully, to search for new informative polymorphic

genetic

markers in plants. It has been called

RAPD mapping,

which is short for randomly ampli-

fied polymorphic DNA. The idea is to amplify as large

a number of bands as

can

be

clearly analyzed by a single electrophoretic lane and then

to compare the patterns

seen in

a diverse set of individuals.

analysis of a number of different genomic targets. This approach is called

multiplex PCR,

and it is carried out in a reverse dot blot format. The procedure is illustrated schematically

in Figure 4.12. Each target to be analyzed is flanked by two specific primers. One is ordi-

nary; the other is tagged with a unique 20-bp overhanging DNA sequence. PCR can be

carried out separately (or in one pot if conditions permit), and then the resulting products

pooled. The tagged PCR products are next hybridized to a filter consisting of

a set of

spots, each of which contains the immobilized complementary sequence of one of

the

Figure 4.12

Inverse dot blot for the multiplex analysis of a

set of

pooled

PCR

reactions.

(a) PCR with a set of

different

tagged

primers, only

one of which

is shown.

(b)Resulting

ADDITIONAL VARIANTS

OF

THE

BASIC

PCR

REACTION

115

For the approach shown in Figure 4.12, it would be far more useful to produce single-

stranded product from the PCR reaction, since only one of the

two strands of the ampli-

fied target can bind to the filter; the other strand will

actually act as a competitor.

Similarly, for DNA sequencing, it is highly desirable to produce a single-stranded DNA

product. Presence of the complementary strand presents an unwanted complication in the

sequencing reactions, and it can also act as a potential

competitor.

To

produce

single

strands in PCR, a very simple approach called

asymmetric PCR

can be

used. Here ordi-

nary PCR is carried out for a few less than the usual number of cycles, say 20. Then one

primer is depleted or eliminated, and the other is allowed to

continue through an addi-

tional 10 cycles of linear PCR. The result is a product that is

almost

entirely

single

stranded. Clearly one can have whichever strand one wants by the appropriate choice of

primer.

For diagnostic testing with PCR, one needs to distinguish different alleles at the DNA

sequence level. A general approach for doing this is illustrated in Figure 4.13. It is called

allele-specific PCR.

In the case shown

in

Figure

4.13, we have a two-allele polymor-

phism. There is a single base difference possible, and we wish to know if a given individ-

ual has two copies of one allele, or the other, or one copy of each. The goal is to distin-

guish among these

three alternatives in a single, definitive test. To do this, two primers are

constructed that

have 3

-ends specific

for

each

of the

alleles. A third general primer is

used somewhere downstream where the two sequences are identical. The key element of

allele-specific PCR is that because

Taq

polymerase

does

not

have a

3

-exonuclease, it

cannot use or degrade a primer with a mispaired 3

-terminus. Thus the allele-specific

primers will only amplify the allele to which they correspond precisely. Analysis of the

results is simplified by using primers that are tagged either by having

different lengths

or

by having different colored fluorescent dyes. With length tagging, electrophoretic analysis

will show different size bands for the two different alleles, and the heterozygote will be

revealed as a doublet. With color tagging, the homozygotes will show, for example, red or

green fluorescence, while the heterozygote will emit

both

red

and

green

fluorescence,

which our eye detects as yellow.

Color can also be used to help monitor the quantitative progress of a PCR reaction.

Colored primers would not be useful unless their color were altered during the chain

extension process. A similar problem holds for the potential

use

of

colored

dpppN’s.

The best approach to date is a slightly complicated strategy, which takes advantage of

the 5 -exonuclease activity of thermostable DNA polymerases (Heid et al. 1996). A

short oligonucleotide probe containing two different fluorescent labels is allowed to

hybridize downstream from one of the PCR primers. The two

dyes

are

close

enough

that the emission spectrum of the pair is altered by fluorescence

resonance

energy

transfer. As the

primer is

extended, the polymerase

reaches

this probe

and

degrades

it.

116

METHODS FOR

IN VITRO

DNA AMPLIFICATION

A color change is produced because the

nucleotide products of the digestion diffuse

too far away from each other for energy transfer to be efficient. Thus each chain exten-

sion results in

the same incremental change in fluorescence. This procedure is called

the TaqMan™ assay. A related spectroscopic trick, called molecular beacons, has re-

cently been described in which a hybridization probe is designed as an oligonucleotide

hairpin with different fluorescent dyes at its 3

-

and 5

-ends (Kramer, 1996). In the

hairpin these are close enough for efficient energy transfer. When the probe hybridizes

to a longer target to form a duplex, its ends

are

now separated far apart

in space, and

the energy transfer is eliminated.

Hairpins can also be used to produce selective PCR amplification. In suppression PCR,

long GC-rich adapters are ligated onto the ends of a mixture

of

target

fragments

(Diatchenko et al., 1996). When the ligation products are melted, the ends of the resulting

single strands can form such stable hairpins

that

these

ends become

unaccessible

for

shorter primers complementary to the adapter sequences. However, if molecules in the

target mixture contain a known internal target sequence, this can be used to initiate PCR.

Chain extension from a primer complementary to the internal sequence will produce a

product with only a single adapter. This will now allow conventional PCR amplification

with one internal primer and one adapter primer.

A final PCR variant is called

DNA shuffling

(Stemmer, 1994). Here the goal is to en-

hance the properties of a target gene product by in vitro recombination. Suppose that a se-

ries of mutant genes exist with different properties; the goal is to combine them in an op-

timal way. The genes are randomly cleaved into fragments, pooled, and

the

resulting

mixture is subjected to PCR amplification using primers flanking the gene. Random as-

sembly of overlapping fragments will lead

to products that can be chain

extended until

full length reassembled genes are produced. These then support exponential PCR amplifi-

cation. The resulting populations of mutants are

cloned and

characterized

by some kind

of screen or selection in order to concentrate those with the desirable properties. This new

method appears to be extremely promising. A very interesting alternative method to shuf-

fle DNA segments uses catalytic RNAs (Mikheeva and Jarrell, 1996).

TOTAL

GENOME

AMPLIFICATION

METHODS

A frequently encountered problem in biological research is insufficient amounts of sam-

ple. If

the sample is a cultured cell or

microorganism, the simplest solution is to

grow

TOTAL GENOME AMPLIFICATION METHODS

117

needed. The issue is how to do this in such a way that the stockpile represents a complete,

or at least a relatively complete and even sampling of the original sample. The danger of

course is that the sample will consist of a set of regions with very different amplification

efficiencies with any particular set of primers. After PCR the stockpile

will

now be

a

highly distorted version of the original, and future experiments will all be plagued by this

distorted view.

One approach to PCR sampling of an entire genome is the method of primer extension

preamplification (PEP). This was designed

to be used on a single cell or

single

sperm.

The detailed rationale for PEP will become apparent when genetics by single sperm PCR

is discussed in Chapter 7. PEP is illustrated schematically in Figure 4.14

a . A mixture of

all possible 4

n primers of length

n is generated by automated oligonucleotide synthesis,

using at each step all four nucleotides rather than just a single one. This extremely com-

plex mixture is then used as a primer. Although the concentration of any one primer is

vanishingly small, there are always enough primers present that any particular DNA seg-

ment has a reasonable chance of amplification. Norman Arnheim and his coworkers have

reported reasonable

success at using this

approach with

n

15 (Arnheim and

Ehrlich,

Figure 4.14

Methods for total genome PCR.

(a) Primer extension preamplification (PEP).

(b)