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

118

METHODS FOR IN VITRO DNA AMPLIFICATION

When we attempted to use the PEP method, we ran into the difficulty that a large num-

ber of template-independent sequences were amplified in the reaction

mixture. This

is

shown

in

the results of Figure 4.15, which illustrates

the

pattern

of hybridization

seen

when

PEP-amplified

S. pombe

chromosome

1

is

hybridized to

an arrayed cosmid

library

providing

a fivefold

coverage of the

S. pombe

genome. Almost

all of

the clones are de-

tected with comparable intensities, even though only about 40% of them should contain

material from chromosome 1. We reasoned that the complex set of long primers might al-

low for very significant levels of primer dimers (Fig. 4.3

b) to be produced, and since the

primers

represented

all possible DNA sequence, their

dimers

would

also

represent

a

Figure 4.15

Examples of ampli-

fication of

S. pombe

chromosome

I DNA.

(a)

A schematic of

an ex-

periment in which directly labeled

chromosome I or amplified chro-

mosome I is used as a hybridiza-

tion probe against an arrayed li-

brary

of the

entire

S.

pombe

genome

cloned into cosmids.

(b)

Actual hybridization results for la-

beled

chromosome

I

(top),

chro-

mosome I labeled after T-PCR

amplification

(center),

chromo-

some I labeled after PEP amplifi-

cation

(bottom).

(From

Grothues

et al., 1993.)

APPLICATION OF PCR TO DETECT MOLECULES OTHER THAN DNA

119

Three variations were introduced to circumvent the primer dimer

problem in

PEP.

Together these constitute an approach we call

T-PCR,

for tagged random primer PCR

(Grothues et al., 1993). Our primers consist of all 4

9 nonanucleotides. Their shorter length

and smaller complexity should be an

advantage compared with the 4

15 compounds used

in ordinary PEP. Each primer was equipped at its 5

-end

with

a constant 17

base

se-

quence; this is the tag. Thus the actual primers used were

GTTTTCCCAGTCACGACN

9

where N is a mixture of A, T, G, and C. After a few rounds of PCR, the resulting mixture

was fractionated by gel filtration, and material small enough to be primer dimers was dis-

carded. Then the remaining mixture was used as a target for amplification

with only

the

tag sequence as a primer (Fig.

4.14

b). As shown

in Figure

4.15,

this

yielded

reaction

products that produced a pattern of hybridization with the

S. pombe

cosmid

array

almost

identical to that seen with directly labeled chromosome 1. Thus we feel that T-PCR offers

very good prospects for uniformly sampling a complex DNA sample. In our hands this

approach has been successful thus far with as little as 10

12 g DNA, which corresponds to

less than a single human cell.

Quite a few variations on this approach have been

developed by

others. One

example

is degenerate oligonucleotide-primed PCR (DOP–PCR) described by Telenius et al.

(1992). Here primers are constructed like

AAGTCGCGGCCGCN

6ATG

with a six

base

totally

degenerate

sequence

flanked

by a

long

5

unique

sequence and

a specific 3 to 6 base unique sequence. The 3

-sequence

serves to select a subset of

potential PCR start points. The degenerate sequence acts to stabilize the primer-template

complex. The

constant

5

-sequence can be used for

efficient amplification

in subsequent

steps just as the tag sequence

is used in T-PCR. It is not

yet

certain how to

optimize

whole genome PCR methods for particular applications. Issues that must be considered

include the overall efficiency of the amplification, the uniformity of the product distribu-

tion, and the fraction of the original target that is present in the final amplified product. A

recently published DOP–PCR protocol (Cheung and Nelson, 1996) looks particularly

promising.

APPLICATION

OF

PCR

TO

DETECT

MOLECULES

OTHER

THAN

DNA

A natural extension of PCR is its use to detect RNA. Two general approaches for doing

this are summarized in Figure 4.16. In one, which is specific

for polyadenylated mRNA,

an oligo-dT primer is used, with reverse transcriptase, to make a DNA copy of the RNA.

Then conventional PCR can be used to amplify that DNA. In the other approach, which is

more general, random (dN)

n ’s are used to prime reverse transcriptase to make initial DNA

copies, and then ordinary PCR ensues.

Less obvious is the use of PCR to detect antigens or other non-nucleic acid molecules.

We originally demonstrated

the feasibility of this approach, which

should have a

broad

APPLICATION OF PCR TO DETECT MOLECULES OTHER THAN DNA

121

Expression vector:

pTSAPA-2

Amino acid residues:

289 per subunit

Subunits:

4 (subunit tetramer)

Molecular mass:

31.4 kDa per subunit

126 kDa per molecule

Biotin binding:

4 per molecule

1 per subunit

IgG binding:

4 per molecule

1 per subunit

(human IgG)

Figure 4.19

Result of detection of tenfold serial dilutions of an antigen, bovine serum albumin,

by immuno-PCR. (Taken from Sano et al., 1992.)

105 times

more sensitive than conventional immunoassays. A major advantage of i-PCR is

that the DNA molecule used is purely arbitrary. It can be

changed at will to prevent the

buildup of

laboratory contaminants. It need not correspond

(indeed it should not corre-

Figur

e

4.20

Isothermal

self-sustained

se-

quence

replication

(3SR).

(a)

Synthesis

of

a

double-stranded

duple

x from

a

starting

RN

A,

using

one

transcript-specific

primer

,

TSP

,

fused

to a sequence

containing

a

promoter

site

(solid

bar)

for

T7

RN

A

polymerase

and

a

second

transcript-specific

primer

B.

(b) Cyclical

amplification

by

transcription

and

con

v e rsion

of

the

resulting

RN

A

mole-

cules to duple

x DN

As.

OTHER ALTERNATIVES TO PCR

125

Figure 4.21

Strand displacement amplification (SDA). A

Hinc

II site with alpha thio DNA deriva-

tives that block

(solid circles)

and do not block

(open circles)

cleavage.

(b)Linear amplification by

strand displacement from one

Hinc

II cleavage site.

(c) Exponential amplification from two

Hinc

II

cleavage sites.

A DNA polymerase I mutant with no 5

-exonuclease activity is used. This leads to

strand displacement. Consider first the effect of this enzyme on the target-primer com-

plex shown in the figure. The primer has a potential

Hinc

II

site

overhanging

the tem-

plate. The polymerase extends both the template and the primer, incorporating alpha-S-

A. The top strand of the resulting duplex can be

cleaved

by

Hinc

II; the

bottom strand

is resistant. This creates a target for

the polymerase

that can strand displace most of

the top strand and continually make

copies

of it,

resulting in linear amplification.

FUTURE OF PCR

127

If they match the target sequence exactly, DNA ligase will efficiently seal them together

provided that a 5

-phosphate is present to form the phosphodiester bond between them. A

complementary set of oligonucleotides can be used to form a ligation product directed by

the other DNA strand. The overall result is to double the number of DNA strands. Both du-

plexes are melted, more oligonucleotides anneal, and the process can be continued indefi-

nitely. With continual thermal cycling, the result is exponential amplification of the target.

This is most easily detected in automated systems by using a capture tag on one of the

oligonucleotides to be ligated and a color-producing tag on the other. This procedure is ob-

viously limited to small DNA target sequences, but it could form a powerful alternative to

allele-specific PCR. Hybrid amplification procedures that combine LCR and polymerase

extension reactions also appear to be very promising.

The final amplification scheme we will discuss is carried out by the enzyme Q

repli-

case. This occurs strictly at the RNA level. Appropriate RNA targets can be made by sub-

cloning DNA samples into vectors that

embed the desired targets within Q

sequences

and place them all downstream from a

T7 RNA polymerase promoter so

that

an RNA

copy can be made to start the Q

replication

process (Q

R). A much more general ap-

proach is to construct two separate RNA probes that can anneal to adjacent sequences on

a target RNA. In the presence of T4 DNA ligase, the two probes will become covalently

joined. Neither prone alone is a substrate for Q

replicase. However, the ligation product

is a substrate and is efficiently amplified (Fig. 4.23).

Q has an unusual mode of replication. No primer is needed. No double-stranded in-

termediate is formed. The enzyme recognizes specific secondary structure features and

sequence elements on the template,

and then makes a complementary copy of it. That

copy dissociates from the template as it is made, and it folds into its own stable secondary

structure which is a complement of that of the template. This structure also can serve as a

template for replication. Thus the overall process continually produces both strands as

targets, much in the manner of a dance in which the two partners

move

frenetically

but

never stay in contact for an extended period.

The usual mode of Q

replication is very efficient. It is not uncommon to make 10

7 to

108 copies of the original target. One can start from the single molecule level. However,

the system is not that easy to manipulate; Q

replicase

itself

is a complex four-subunit

enzyme not that commonly available. The

procedures needed to prepare the DNA target

for Q replication are somewhat

elaborate, and there are considerable restrictions

on

what sorts of RNA insertions can be tolerated by the polymerase. For all these reasons the

Q amplification system is most unlikely to replace PCR as a general tool

for DNA

analysis. It may, however, find unique niches for analyses where the idiosyncrasies of the

system do not interfere, and where very high levels of amplification at constant tempera-

ture are needed.

FUTURE OF PCR

In this chapter we have illustrated myriad variations and potential applications of PCR. In

viewing these, it is important to keep in mind that PCR is a young technique. It is by no

means clear that today’s versions are

the optimal ones or the most easily adaptable ones

for the large-scale automation eventually needed for high-throughput genome analysis.

Much additional thought needs to be

given on how best to format PCR for widespread

use and how to eliminate many of the

current glitches and irreproducibility

inherent in

such a high-gain amplification system.