Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 14

480

SEQUENCE-SPECIFIC MANIPULATION OF DNA

SEQUENCE-SPECIFIC DNA CAPTURE

In the majority of cases, the motivation behind attempts to develop ways for sequence-

specific DNA cleavage is to provide access to particular

DNA sequences, as just de-

scribed above for RARE cleavage. A more direct approach would be to develop methods

for the specific capture of DNA sequences of particular interest. There are a number of

different approaches that have been used to purify DNAs based on sequence rather than

size. Biological methods we have already described include PCR or differential cloning.

These are extremely effective, but because the steps are

usually carried out in solution

and involve a number of enzymes, the targets of such purifications are usually limited in

size. Large insert cloning systems have been described, but these are not yet applicable to

differential cloning. For example, YAC cloning can be used

to immortalize a set

of large

DNA fragments, but then this set has to be screened to identify the clone containing a

particular sequence of interest. Such screening can be quite time-consuming. Instead of

screening, we are concerned here with methods that

select the target of interest.

Purification techniques are a natural way to select molecules with desired properties such

as specific DNA sequences.

In principle, a DNA sequence could be selected by cloning, by PCR, by binding to a

specific protein, by binding to the complementary strand to

form a duplex, or by binding

to a third strand to form a triplex. In each of these approaches the desire is a simple se-

quence-specific purification method. In this section we will limit our attention to physical

purifications based solely on DNA base interactions. Such

methods have been called in

the past, perhaps somewhat inelegantly, DNA fishing. Differential PCR and cloning meth-

ods will be described later in the chapter. The advantage of the pure, physical methods is

that if successful, they are very easy to implement and easy

to scale up to large

numbers

of samples or large quantities. A key obstacle in the use of

such methods

is that

once the

DNA target is captured by a probe, there either must be an

efficient way to release it, or

one has to have an efficient way of working directly with

the immobilized DNA (as in

solid state DNA sequencing, described earlier in Chapter 9).

TRIPLEX-MEDIATED DNA CAPTURE

The major advantage in using a third strand to capture a

DNA duplex is that there is no

need to melt the duplex. This avoids potential damage arising from any preexisting

nicks

in the DNA strands. It also helps avoid many of the complications caused by the extensive

interspersed repeats in the DNA of higher organisms. There are a number of potential ap-

plications for triplex capture of DNA. For example, triplex formation could be used, in

principle, to isolate from a library all those clones that contained specific homopurine-ho-

mopyrimidine sequences such as (AG)

n , (AAG)

n , or (AAAG)

n . These clones are all poten-

tially useful genetic probes, since all are VNTRs. Such clones have been isolated in the

past by screening libraries by hybridization

with the repeated seqeunce. However,

if one

is

interested in a large set of such clones,

each one detected by hybridization

has to

be

TRIPLEX-MEDIATED

DNA

CAPTURE

481

TABLE 14.1

Repeating Single Sequence Motifs in the Genbank Database

(1991)

Dinucleotide

AG

24

AT

18

AC

86

TG

1

Trinucleotide

AAC

14

CCG

12

AGG

9

AAT

7

AGC

10

AAG

3

ATC

2

ACC

2

Tetranucleotide

AAAG

20

AGCC 1

AACT

1

AAAT

22

AGAT

9

ACAT

1

ACAG

3

ATAG

3

ACGC

1

ACAT

2

AGCG

1

ACTG

2

AAGG

8

ATCC

8

AGGG 1

AAGG

13

AATC

4

AGCT 1

Lohger

AAAAG

1

AAAAAAAAAG

1

TTTTTG

1

Two other potential

applications for triplex capture concern the purification of a spe-

cific DNA sequence. If a genomic fragment contains a known specific homopurine se-

quence,

one should be

able to capture this sequence away from the entire genome by

triplex formation on a solid support. Alternatively, such a sequence could be introduced

into a desired section of a genome to allow subsequent capture of DNA from this region.

Such approaches, once they have matured, could potentially expedite the analysis of se-

lected

megabase regions

of DNA considerably. The second potential use of triplex cap-

removed in advance.

A number

of

different ways to use triplexes to purify DNA have

been

explored.

Although

much

more

development work needs to be done, the preliminary findings

are

very promising. In our work, magnetic microbeads were used as the solid support. The

power of this general approach was described earlier in applications for solid state DNA

sequencing. Magnetic microbeads are available commercially that are coated with strep-

tavidin.

Streptavidin

will bind specifically to virtually any biotinylated

macromolecule.

Any desired small

DNA

sequence can be prepared with a 5

-biotin by the use of biotin

482

SEQUENCE-SPECIFIC MANIPULATION OF DNA

phosphoramidite in ordinary automated oligonucleotide

synthesis. Longer 5

-biotinylated

probes can be made by PCR using 5

-biotinylated primers. Alternatively, internally bi-

otinylated

DNA sequences can be made by PCR

using biotinylated base analogs as

dpppN substrates. Some of these compounds are incorporated quite efficiently by DNA

polymerases. Once attached to the streptavidin microbeads, biotinylated DNA probes are

still quite accessible for hybridization or for triplex formation. A schematic illustration of

the way microbeads have been used to develop a simple triplex capture method of DNA

purification is shown in Figure 14.13. The key

point is that a permanent magnet can be

used to hold the beads in place while supernatant is removed and exchanged. This allows

very rapid and efficient exchanges of reagents. At pH 5 or 6 any duplex DNA captured by

the complementary sequence on a bead will remain attached. At pH 8 the captured DNA

will be readily released and removed with the supernatant.

Three different experiments have been used to

test the ease and efficiency of

triplex

DNA capture with magnetic microbeads. In the first,

an artificial mixture was made of

two plasmids. One contained just a vector with no known forming capability, which in-

cluded a lacZ gene. When this plasmid is transformed into

E. coli,

blue colonies are pro-

duced on the appropriate indicator plates, which contain a substrate for the beta-galactosi-

dase product of this gene that yields a blue-colored product. The second plasmid, initially

Figure 14.14

Outlined of the procedure used to purify (AG)

n -containing clones from a chro-

TRIPLEX-MEDIATED DNA CAPTURE

485

rimidine

sequence

in

yeast

(S.

cerevisiae)

chromosome III. This strain was originally

made to test the efficiency of triplex-mediated DNA cleavage. We were able to obtain and

use the same strain to check the efficiency of triplex-mediated DNA capture. Instead of

working with yeast genomic DNA, which would suffer considerable shear damage under

the conditions used to manipulate the magnetic microbeads, we first subcloned this strain

of yeast into a plasmid vector. Then plasmid DNAs were selected using magnetic mi-

crobeads containing the appropriate homopyrimidine sequence. In practice, 50% of the

clones isolated contained the correct DNA insert as determined by DNA sequencing. The

other contaminating clones had a similar, but not identical, sequence that was a natural

component

of the

yeast

genome. Presumably

this

contaminant could

have been selected

AFFINITY

CAPTURE ELECTROPHORESIS

487

To avoid the problems with the currently available form of triplex-mediated ACE, an

alternative capture scheme was developed that could be implemented at neutral pH. This

scheme lacks the attractive generality of triplex

capture because it is applicable only to

the ends of DNA fragments, and it requires that information be available about the DNA

sequence at

one end, or at least

that a clone

be available

that overlaps

the

ends

of the

DNA to be captured. This is a major limitation, in principle, but it is less confining in

practice. A major potential application for ACE is to try to purify specific large restriction

fragments from complex mixtures. If a linking library that

corresponds to

the

ends

of

these fragments is available, as described in Chapter 8, then the necessary clones or se-

quences will already be in hand.

The basic scheme of an alternative ACE procedure, termed end-capture electrophore-

sis, is shown in Figure 14.18. In this scheme, the ends of a long DNA duplex are treated

with an exonuclease like

E. coli

Exonuclease III, or DNA

polymerase

in the absence of

triphosphates, to remove a small portion of the 3

-ends of the duplex and expose the com-

plementary 5

sequence as single strand. The affinity capture medium is made, as before,

with the sequence complementary to the target. In this case the beads will contain the au-

thentic 3

-end of the large DNA fragment, synthesized from known sequence or isolated

as the appropriate half of a linking clone. Capture consists of ordinary duplex formation.

The challenge is to find an efficient and nondisruptive way to release the target after it has

been captured. An effective way to do this, illustrated in Figure 14.19, is to prepare the

capture probe so that it contains the base dU

instead of T. This still allows efficient,

sequence-specific strand capture. However, subsequent treatment with Uracil DNA gly-

cosidase, an enzyme that participates in the repair of DNA (Chapter 1), will release the

captured target. The same general idea was described in Chapter 4 as

a

method

for

minimizing PCR contamination.