Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 11

Primer length

10

11

12

Unique

0.918

0.979

0.998

DNA synthesis.

The first multiplex synthesizers built could only make 4 to 10 samples at

a time; this

is far less than would be needed for large-scale DNA sequencing by primer

walking. Recently

instruments

have been described that can make 100 primers at a time

in amounts 10%

or less of

what is now done conventionally. However, such instruments

are not yet widely available. In the near

future primers will be made thousands at a time

by in situ synthesis in an array format (see

Chapter 12).

A second major problem with directed

sequencing is that each sample must be done

walking.

Instead, one must

work with at least as many samples as there are

DNA

se-

quencing

lanes

available,

and ideally one would be processing two to three

times

this

amount so

that

synthesis

is

never a bottleneck. This means that the whole project has to

be orchestrated carefully

so

that an efficient flow of samples occurs. Directed

sequencing

PRIMING WITH MIXTURES OF SHORT OLIGONUCLEOTIDES

365

shotgunning serves to accumulate large numbers of samples, which are then extended by

parallel directed sequencing.

Three practical considerations enter the

selection

of

particular primer

sequences.

These can be dealt with by existing software packages that scan the sequence and suggest

potentially good primers. The primer must be as specific as possible for the template.

Ideally a sequence is chosen where slight mismatches elsewhere on the template are not

possible. The primer sequence must occur once and only once in the template. Finally the

primer should not be capable of forming stable secondary structure with itself—intramol-

ecularly, as a hairpin, or intermolecularly—as a dimer. The former category is particularly

troublesome, since, as illustrated in Chapter 10, there are occasional DNA sequences that

form unexpectedly stable hairpins, and we do not yet understand these well enough to

generalize and be able to identify and reject them a priori.

PRIMING WITH MIXTURES OF SHORT OLIGONUCLEOTIDES

Several groups have recently

developed schemes

to circumvent the difficulties required

by custom synthesis of large numbers of unique DNA sequencing primers. The relative

merits of these different schemes are still not

fully evaluated, but on the surface, they

seem quite powerful. The first notion, put forth by William Studier, was to select, arbitrar-

ily, a subset of the 4

9 possible 9-mers

and make all of these compounds in advance. The

chances that a particular 9-mer would be present in a 45-kb cosmid are given below, cal-

culated from the Poisson distribution P(

n ), where

n

is the expected number of

occur-

rences:

P(O) 0.709

P(1) 0.244

P(

1) 0.047

Therefore, with no prior knowledge about the

sequence,

a

randomly

selected

9-mer

primer will give a readable sequence about 20% of the time. If it does, as shown in Figure

11.4, its complement will also give readable sequence. Thus the original subset of 4

9

compounds should be constructed of 9-mers and their complements.

One advantage of this approach is that no sequence information about the sample is

needed initially. The same set of primers will be used over and over again. As sequence

data accumulate, some will contain sites that allow

priming

from members

of the

pre-

made 9-mer subset. Actual simulations indicate that the whole process could be rather ef-

ficient. It is also parallelizable in an unlimited fashion because one can carry as many dif-

ferent targets along as one has sequencing gel lanes

available. An obvious and

serious

tive solution to this problem

was put forth by Wacslaw Szybalski. His original scheme

was to make all possible 6-mers

using current automated DNA synthesis techniques. With

current methods this is quite

a manageable task. Keeping track of 4096 similar com-

rected sequencing by primer walking. After each step the new sequence

must

be

scanned

to determine which combination of 6-mers should

be selected as the

optimal

choice

for

the next primer construction.

The early attempts to carry out tandem

6-mer priming seemed

discouraging. The

method appeared to be noisy and unreliable. Too

many of the lanes

gave unreadable

se-

PRIMING WITH MIXTURES OF SHORT OLIGONUCLEOTIDES

367

likely to perform slightly better than ligated mixtures of smaller compounds because the

possibility of using the 6-mers in unintended orders, or using only two of the three, is

avoided. There is enough experience with longer primers to know how to design them to

minimize chances of failure. Similar experience must be gained with 6-mers before an ac-

curate evaluation of their relative effectiveness can be made.

Two more recent attempts to use 6-base oligonucleotide mixtures as primers avoid the

ligation step. This saves time, and it also saves any potential complexities arising from

ligase side reactions. Studier’s approach is to add the 6-mers to DNA in the presence of a

nearly

saturating

amount

of

single-stranded DNA binding protein (ssb). This protein

binds to DNA single strands selectively and cooperatively. Its effect on the binding of sets

of small complementary short

oligonucleotides is shown schematically in Figure 11.6.

The key point is the high cooperativity

of ssb to single-stranded DNA. What this does is

to make configurations in which 6-mers are bound at separate sites less favorable, ener-

getically, than configurations in which they are stacked together. In essence, ssb coopera-

tivity is translated into 6-mer DNA binding cooperativity. This cuts down on false starts

from individual or

mispaired

6-mers. An unresolved issue is how much mispairing still

can go on under the conditions used. We have faced before the problem in evaluating this;

there are many possible 6-mers,

and many

more possible sets of 6-mer mixtures, so only

a minute fraction of these possibilities has yet been tried experimentally. One simplifica-

tion is the use of primers with 5-mC and 2-aminoA, instead of C and A, respectively. This

allows five base primers to be substituted for six base primers, reducing the complexity of

the overall set of compounds needed to only 1024.

An

alternative approach

to

the problem has been developed by Levy Ulanovsky. He

has shown that conditions can

be

found where it is possible to eliminate both the ligase

and the ssb and still see highly

reliable priming. Depending on the particular sequence,

some five base primers can be substituted for six base primers. The effectiveness of the

approach appears to derive from the intrinsic preference of DNA polymerase for long

primers, so a stacked set is preferentially utilized even though individual members of the

set may bind to the DNA template

elsewhere alone or in pairs. It remains to be seen, as

each of

these approaches

is developed further, which will prove to be the most efficient

and accurate. Note that in any directed priming scheme, for a 40-kb cosmid, one must se-

quence 80 kb of DNA. If a net of 400 new bases of high-quality DNA sequence can be

completed per run, it will take 200 steps

to

complete the walk. As a worst-case analysis,

one may have to search the 3

-terminal 100 bases of a new sequence to find a target suit-

able for priming. Around

500

base pairs

of

sequence must actually be accumulated per

TRANSPOSON-MEDIATED DNA SEQUENCING

369

Figure 11.7

Structure of a typical transposon useful for DNA sequencing.

N is a Not I site;

a and

b are unique transposon sequences. Arrow heads show the terminal inverted repeats of the transpo-

son.

coli.

It is present in an

original vector containing a single infrequent

cutting site

(which

can be the same as the ones

on the transposon) and two unique known DNA sequences

near to the vector-insert boundary. The transposon may jump into the cloned DNA. If it

does so, this appears to occur approximately at random. The nonselectivity of transposon

hopping is a key assumption

in the potential effectiveness of this strategy. From results

that have been obtained thus far, the randomness of transposon insertions seems quite suf-

ficient to make this approach viable.

DNA from cells in which transposon jumping has potentially occurred can be trans-

ferred to a new host cell that does not have a resident transposon, and recovered by selec-

tion. The transfer can be done by mating or by transformation. Since the transposon car-

ries a selectable marker, and the vector

which originally contained the

clone carries

a

second selectable marker, there will be no difficulty in capturing the desired transposon

insertions. The goal now is to find a large set of different insertions and map them within

the

original cloned

insert

as efficiently as possible. The infrequent cutting sites placed

into

the

transposon

and the

vector greatly

facilitate this analysis. DNA is

prepared from

Figure 11.8

One strategy for DNA sequencing by transposon hopping.

a, b, and N are as defined

Figure 11.9

DNA sequencing from a

set of transposon insertions.

(a ) Chosing a set of properly

spaced transposons. (

b ) Use of

transposon sequences as primers.

Now the position of the transposon insertion is known. One can select samples that ex-

tend through the target at intervals than

can be spanned by sequencing,

as

shown

in

Figure 11.9. To actually determine the sequence, one can use the two unique sequences

within

the transposon as primers. These will always allow sequence to be

obtained

in

both directions extending out from the site of the transposon insertion (Fig. 11.9). One

does not a priori know the orientation of the two sequences primed from the transposon

relative to the initial clone. However, this

can be determined, if necessary, by

reprobing

the infrequent-cutter digest fingerprint of the clone with one of the transposon unique se-

quences. In practice, though, if a sufficiently dense set of transposon inserts is selected,

there will be sufficient sequence overlap to assemble the sequence and confirm the origi-

nal map of the transposon insertion sites.

DELTA

RESTRICTION

CLONING

This

is a relatively straightforward procedure that allows DNA sequences

from

vector

arms to be used multiple times as sequencing primers. It is a particularly efficient strategy

for sampling the DNA sequence of a target in numerous places and for subsequent, paral-

lel, directed primer sequencing. Delta restriction cloning requires that the target of inter-

est be contained near a multicloning site in the vector, as shown in Figure 11.10. The var-

ious enzyme recognition sequences in that site are used, singly, to digest the clone, and all

of the resulting digests are analyzed by gel

electrophoresis. One looks for

enzymes

that

cut the clone into two or more fragments. These enzymes will have cutting

sites within

the insert. From the size of the fragments

generated, one can determine approximately

where in the insert the internal cleavage site is located. Those cuts that occur near regions

of the insert where sequence is desired can be converted to targets for sequencing by di-

luting the cut clones, religating them, and transforming them back into

E. coli.

The effect

of the cleavage of religation is to drop out from the clone all of the DNA between the

multicloning site

and the internal restriction

site. This brings the vector arm containing