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

500

SEQUENCE-SPECIFIC MANIPULATION OF DNA

than the true target, the ratio of true target to mimic products can be determined. These

approaches

fall

far

short

of the mark

when the goal

is

to

analyze many

genes

of

interest.

A number of interesting methods have been developed to sample mRNAs or correspond-

ing cDNAs and look for expression differences or patterns in biological states. These are

still evolving rapidly.

In differential display a degenerate short PCR primer is used to amplify the cellular

population of cDNAs. This can be a poly dT complementary to the 3

poly A sequence of

messages, it can be an anchored dT

n

(Liang

et

al.,

1994), a short oligonucleotide, or a

short sequence complementary to the end of DNA fragments generated by type II-S re-

striction enzymecleavage (Kato, 1995). Type II-S enzymes cut outside of their recogni-

tion sequences to yield a mixture of different single-stranded overhangs. A subset of this

mixture can be captured by ligation to complementary

overhangs,

a technique

that

has

been called molecular indexing (Unrau and Deugau, 1994). Regardless of the method

used, the result is to reduce the complexity of the mRNA population and amplify the re-

sulting cDNA to produce a discrete set of species to analyze quantitatively. The analysis

can be performed by conventional or fluorescence-detected gel electrophoresis, or by hy-

bridization (or two-color competitive hybridization) to an array of DNA probes analogous

to the arrays used for SBH (Chapter 12).

These differential display methods are extremely

powerful

and

broadly

applicable.

They suffer from a common limitation that whenever PCR amplification must be applied

to a complex sample, the actual population of preexisting mRNAs will be distorted by

differences in their ability to sustain multiple rounds of amplification. Two very different

approaches for avoiding PCR-induced distortions are

actively

being

explored. Picking

cDNA clones at random and identifying them after sequencing is a

powerful but

expen-

sive method. Alternatively, cDNAs can be sampled prior to amplification by cutting out a

specific short fragment. The fragments are co-ligated into concatemers and amplified as a

group. Then sequencing of cloned concatemers reveals relative abundances in a clever ap-

proach termed serial analyses of gene expression (SAGE).

An alternative to differential display is differential cloning. Differential cDNA cloning

can proceed by schemes very similar to the one shown in Figure 14.25, or they

can in-

volve more

elaborate techniques such as illustrated

for

the

preparation

of

normalized

cDNA libraries in Chapter 11. The objective is to recover cDNAs that represent messages

present in one cell type but not another. The more carefully the cell types are selected, to

differ just in the desired characteristics, the more efficient will be the search for the genes

responsible for those characteristics. Differential cDNA cloning can be used to prepare

genes specific for particular tissue types, developmental stages,

chromosome

origin, or

even subchromosomal origin. These procedures are particularly effective when the target

differences are very small and precisely defined. Examples are differences between unac-

tivated and activated lymphocytes, to recover specific immune response genes, or differ-

ences between regenerating and nonregenerating tissue to

isolate

specific

growth

factors.

As in genomic subtractive cDNA cloning, PCR can be used very effectively to solve most

problems

caused

by

small

samples

or

rare messages.

In

principle,

PCR

should

allow

cDNAs to be made and subtracted from targets as small as single cells.

COINCIDENCE

CLONING

Subtractive cloning allows the selective isolation of DNAs that differ in two samples. A

related,

but technically

somewhat

more demanding

approach

is

coincidence

amplifica-

COINCIDENCE CLONING

505

BOX 14.3

(Continued)

Figure 14.33

How coincidence cloning might be used to enrich the DNA from particular large

re-

striction

fragments

seen

by hybridization in rodent-human hybrid cells.

(a) Restriction map of the

region;

X and

Y

indicate two human-specific probes.

(b) Southern

blot of a PFG fractionation hy-

bridized

with the

two available probes. Intact material from a duplicate

PFG run would be used to

HUMAN INTERSPERSED REPEATED DNA SEQUENCES

Most repeated sequence DNA in the human genome and other complex genomes appears

to be interspersed with single-copy

DNA. Although plenty of tandem repeats

exist, such

as VNTRs, except for centromeres these actually make up only a small

fraction of

the

class of highly repeated sequences. The original demonstration that the bulk of the human

repeats was interspersed was done

in a series of classic experiments

by Britten

and

shown in Figure 14.34

a . A trace amount of labeled total human DNA is sheared randomly

to various average lengths,

L . This DNA is hybridized in solution with a vast excess of

much shorter driver DNA. The driver DNA consists of unlabeled total human DNA iso-

lated as all of the duplex that

forms at

a

C 0 t of less than or equal to 50. This sample will

contain all significant high-copy number human repeats but will contain very little single-

copy DNA or infrequent repeats such as those seen in gene families. The hybridization of

the driver with the labeled DNA is carried out at a

C 0 t of 12.5. The goal of the experiment

is to determine how much of the total labeled DNA can be captured by the repeated DNA

driver during the hybridization.

The basic idea behind the experiment in Figure 14.34

a is that all the DNA in clustered

repeats will be captured very easily and efficiently, regardless of the length of DNA target

used. In contrast, when

L

is small, most interspersed DNA will be captured without flank-

ing single-copy sequences. As

L increases,

more and more single-copy DNA will be cap-

HUMAN INTERSPERSED REPEATED DNA SEQUENCES

507

Figure 14.34

Analysis of

the pattern of repeats in

human

DNA by

renaturation kinetics.

(a)

Design of the basic experiment. Purified repeat is used in excess

as the

driver

to capture indexed

genomic DNA fragments of length

L . (b) Two possible extreme patterns of repeating sequence.

tured by virtue of its neighboring repeated DNA (Figure 14.34

b ). A typical experimental

result from such a procedure is shown in Figure 14.35. It is evident

that

only

about a

quarter of the genome is captured when

L

is small;

this provides an estimate for the total

amount of highly repeated DNA. Almost all of the genome is captured by the time the av-

erage fragment size reaches 8 kb. Thus

many single-copy DNAs have a

repeated

se-

quence within 1 kb, and almost all single-copy DNAs have a repeat within 8 kb. The sim-

plest fit to the data in Figure 14.35

suggests the occurrence of an

interspersed

repeat

every 3 kb. A more elaborate and more accurate fitting procedure suggests that the distri-

bution of repeats is bimodal. About 58%

of the genome has a

repeat on

average

every

1 kb; the remainder of the genome has a repeat on average every 8 kb. Of course this

analysis does not reveal any information about the nature of the repeats or the number of

different basic kinds of repeats and their particular distribution.

In fact there are just a few known major types of interspersed repeats in the

human

genome. Their properties are summarized in Table 14.3. By far the most common human

repeat is a sequence called Alu because it contains two cutting sites for the restriction en-

zyme Alu I. Thus when human DNA is cut with Alu I and fractionated by size, the result-

ing material shows a bright, specific size

band standing out from a background

broad

smear of other fragment sizes. There may be as many as 10

6 Alu sequences in the human

genome. Very similar sequences are seen in other primates, but in more diverged species,

like rodents, the Alu-like sequences are sufficiently different

that Alu’s

can be

used

as

ver, as a function of the length,

L , of the genomic DNA used. Adapted from Moyzis et al. (1989).

DISTRIBUTION OF REPEATS ALONG CHROMOSOMES

509

Figure

14.37

DNA rearrangements produced by recombination between interspersed repeats like

the Alu

sequence.

(a) A direct repeat leads to a DNA deletion because the short fragment produced

is unlikely to be

retained or replicated.

(b) An inverted repeat leads to an inversion in the DNA se-

quence.

The next most common human repeat is the L1 sequence. It sometimes called Kpn be-

cause it has characteristic restriction sites for the enzyme Kpn I. The Kpn family is quite

complex. The total sequence is about 6.4 kb in length. However, only 10% to 20% of the

repeats are full length. Most others retain only the 3

-terminal kb of the L1. These 3

L1’s

occur at about a tenth the frequency of the Alu sequence. L1’s are

more similar in pri-

mates and rodents than Alu’s.

Thus they must be used cautiously

as

species-specific

probes. L1 sequences are an example of the class of repeats called long interspersed re-

peating sequences or LINES. Altogether the repeated sequences listed

in

Table

14.3 add

up at most to 350 Mb of DNA. Centromeric satellite is estimated to account for another

150 to 300 Mb of DNA. This material is tandemly repeating (Chapter 2). Thus at least

100 Mb of the total estimated 750 Mb (25%) of repeated human DNA remains unac-

counted for, and we may be missing as much half of all the repeated sequences.

Interspersed repeats appear to act as recombination hot spots. The sequence of most

examples of Alu repeats is similar enough that recombination between pairs of such re-

peats is sometimes seen as a cause of disease alleles. As shown in Figure 14.37, depend-

ing on whether the repeats are head to head (tandem) or head to tail (inverted), the result

of an intrachromosomal recombination event will be an inversion

or

a

deletion.

Recombinations between repeats on different chromosomes will produce translocations.

The low-density lipoprotein receptor is one of the genes where a deletion caused by inter-

Alu recombination has been seen. In this case the result is to produce a disease allele for

familial hypercholesterolemia.

DISTRIBUTION OF

REPEATS

ALONG CHROMOSOMES

In Chapter 2 we illustrated the fact that light bands and dark bands

of human

chromo-

somes had some very different general properties. A commonly accepted mechanism for

the spread of interspersed repeats is that at least some of the copies of these sequences are

mobile elements and

can spread

themselves or copies to other sites. This

has

definitely