Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 13

DIAGNOSTICS AT

THE DNA

LEVEL

453

TABLE 13.3 Forty-Three Somatic Mutations of Colorectal Tumors in the APC Gene

C113(F)

142

a

a tag/CTG

: atag/CTG

Splice acceptor

C31, C124(F)

213

CGA

: T

GA

Arg

: Stop

C24

279

aatttttag/GGT

: agtttttag/GGT

Splice acceptor

C47

298

C

ACTC

: CTC

AC deletion

C108(F)

302

CGA

: T

GA

Arg

: Stop

C135

438

CAA/gtaa

: CAA/g

c aa

Splice donor

C33

516

AAG/gt

: AAG/ tt

Splice donor

C28

534

AAA

: T

AA

Lys

: Stop

C10

540

TTA

: TT

AT

A insertion

C37

906

T

C TG

: TTG

C deletion

A128(F)

911

GAA

: G

G

A

Glu

: Gly

C23

1068

TCAAGGA

: GGA

TCAA deletion

C11, C15

1114

CGA

: T

GA

Arg

: Stop

C20

1286

GAA

: T

AA

Glu

: Stop

A53

1287

ATA

: A

ATA

A insertion

C27

1293

ACACAGGAAGCAGATTCT

31 bp deletion

GCTAATACCCTGC

AAA

: AAA

C7, C21

1309

G

AAAAGAT

: GAT

AAAGA deletion

C14

1309

GAA

: T

AA

Glu

: Stop

A41

1313

ACT

: G

CT

Thr

: Ala

C31, C42

1315

TCA

: T A

Ser

: Stop

A44

1338

CAG

: T

AG

Gln

: Stop

C22

1353

GAATTTTCTTC

: TTC

8 bp deletion

A56

1356

TCA

: T G A

Ser

: Stop

C4, C27

1367

CAG

: T

AG

Gln

: Stop

C10

1398

AG TCG

: TCG

AG deletion

C19

1398

AG

T C

: AGC

T deletion

A43

1411

A

G TG

: ATG

G deletion

C16

1420

CC

C A

: CCA

C deletion

C40, A52(F)

1429

GAA

: T

AA

Gln

: Stop

C29

1439

C

C TC

: CTC

C deletion

C37

1446

G

CTCAAACCAAGC

: GGC

10 bp deletion

A50(F)

1448

T

T AT

: TAT

T deletion

A49(F)

1465

AG TGG

: TGG

AG deletion

C23

1490

C

ATT

: CTT

A deletion

C12

1492

GC

C A

: GCA

C deletion

A41

1493

ACAGAAAGTAC TCC

: TCC

11 bp deletion

C3

1513

GAG

: T

GAG

T insertion

454

FINDING GENES

AND MUTATIONS

TABLE

13.4 Summary of Sequence Change in 260 Consecutive Cases of Hemophilia B

Number

Percentage

1. Number with sequence changes in the eight regions of likely

249

96

functional significance

2. Of those with sequence changes, number of independent

182

73

mutations

a

3. Of independent mutations, number with a second sequence

6

3

change

4. Type of independent mutation:

Transitions at CpG

48

26

Transitions at non-CpG

65

36

Transversions at CpG

8

4

Transversions at non-CpG

35

19

Small deletions and insertions (

50 bp)

15

8

Large deletions (

50 bp)

10

6

Large insertions

1

0.6

5. Location of independent mutations:

b

Promoter

1

0.5

Coding sequence

c

163

86

Splice junctions

12

6

Intron sequences away from splice junctions

1

0.5

Poly A region

0

—

Unlocalized (total gene deletions)

5

3

Unknown

b

8

4

6. Functional consequences of observed independent mutations

Protein with amino acid substitutions

114

63

Garbled protein (truncated, frameshifted or partial or full

54

30

deletion of amino acids)

Abnormal splicing

13

7

Decrease expression

1

0.6

Source:

Adapted from Sommer et al. (1992).

a Recurrent mutations were judged independent if the

haplotypes differed. In a

few cases recurrent

mutations

with the same haplotype were judged independent because the origin of mutation was determined. In four pa-

tients recurrent mutations were judged independent because the races of the individuals were different.

minimal test would be to proceed

exactly as described above, except that now, presence

of

heteroduplex

indicates

that

the

individual

is a carrier, unaffected for the disease.

However, this

test would

be ambiguous because

absence of heteroduplex would imply

that

either the

individual

in question is a homozygous normal or a homozygous affected.

An additional test is required to resolve this

ambiguity. This second test is designed as

shown in Figure

13.14

c . DNA from

the individual to be tested is mixed with a standard

DNA sample from a person previously shown to be normal, not a carrier. If the test indi-

vidual carries the disease, four DNA products will be formed; two of these will be het-

eroduplexes. If

the test individual is

normal, no heteroduplex products will be produced.

methods for genetic testing must be noted. Successful heteroduplex detection will find

any DNA alterations, whether or not these are disease alleles or

harmless polymorphisms.

Thus

what

heteroduplex detection does is indicate the presence

of a DNA sequence vari-

ant.

Once

this is found, DNA sequencing will frequently be needed to examine the char-

Figure 13.15

A single site mutation will serve to generate the formation of two different

heteroduplexes.

HETERODUPLEX DETECTION

457

gives a different and discrete set of heteroduplexes. Thus a

test that detected a specific

half of the possible heteroduplexes would suffice:

M UTATION

H ETERODUPLEXES

A–T to T–A

A–A and T–T

G–C to C–G

G–G and C–C

A–T to G–C

G–T and A–C

A–T to C–G

A–G and C–T

becomes rather larger. There are four possible single base

insertions

or deletions, but

these are likely to be susceptible to complexities caused by

the local

sequence

context.

For example, in the sequence shown below, two altered mismatched structures compete

with each other.

A–T

A–T

A–T

A–T

G–C

C

G–C

G–C

9: G–C

;: G–C

C ;9 G–C

T–A

T–A

T–A

T–A

Much

more work needs to be done to

characterize the properties of such structures in

more

detail. This will have to be

done before the overall accuracy of any

proposed

method of heteroduplex detection can be validated for clinical use.

At least six different basic methods for detecting heteroduplex DNA have been de-

scribed. All of these tests work well

in some cases. However, none have yet been

proved

large

a

DNA target can be examined directly. The larger the target, the fewer fragments

will

be

needed to cover

a

whole gene. However, if targets are too large,

they

may

have

such

a

high probability

of

containing a phenotypically silent polymorphism

that

the

ad-

Figure

13.16

Four different methods

for

direct detection of heteroduplex DNA. (

a )

Nicking with

S1 nuclease. (

b ) Trimming and nicking an RNA-DNA hybrid with ribonuclease. (

c ) Reaction of un-

paired

bases

with

organic

molecules

like carbodiimides. (

d ) Binding

of

mut S protein to

the site of

the mismatch.

mismatched

bases

in a heteroduplex (Fig. 13.16

a ). Then the

resulting

shortened DNA

fragment could be detected by a Southern blot or by PCR. The difficulty with this ap-

proach is that no known combination of enzyme and reaction conditions allows reliable

cleavage at single base mismatches. Larger mispaired targets are needed before cleavage

becomes efficient and selective. Other nucleases that look very promising for such studies

include bacteriophage T4 endonuclease VII.

RNase nicking can be used as an alternative to DNase nicking. Here a single strand of

the DNA to be tested is annealed with RNA made from a DNA sample representing the

normal allele (Fig. 13.16

b ). This is easily accomplished, for example, by subcloning that

allele downstream from a strong in vitro promoter like that for T7 RNA polymerase. One

can produce either internally labeled RNA,

and

look for

two shorter fragments as a sign

of fragmentation,

or end-labeled RNA, and look for one

shorter fragment. In this latter

case it is necessary to have the test DNA extend beyond the labeled end of the RNA; oth-

erwise, the RNase will remove the label. Some workers swear by the reliability and sensi-

tivity of the RNase approach, but many others have apparently been unable to use it suc-

cessfully.

Chemical methods can be used instead of enzymes to mark or to cleave at the site of a

mismatch. A particularly effective approach has been the use of water-soluble carbodi-

imides

which can

react

with mismatched T

or

G (Fig.

13.16

c ). These compounds

are

HETERODUPLEX

DETECTION

459

available radiolabeled, so one can detect the presence of a mismatch by the incorporation

of radioisotope. Alternatively, there are monoclonal antibodies available that are specific

for the carbodiimide reaction product. This is a very powerful analytical tool, since it al-

lows physical fractionation of any heteroduplexes, which are thus purified and concen-

trated if needed for subsequent analysis. The carbodiimide approach does have two dis-

tinct disadvantages. It cannot detect all possible

mismatches, and it has

been

reported

to

be a difficult test to master in the laboratory.

The fourth test based on protein recognition of heteroduplexes is rather different be-

cause it does not involve enzymatic cleavage. In

E. coli,

a protein is made by

the

mut S

gene that recognizes mismatches and binds to them (Fig. 13.16

d ). This is an early step in

the excision and repair of mismatched bases. The binding of

mut S protein can be detected

in a number of different ways. DNA, once bound by

mut S, will stick to nitrocellulose fil-

ters, while free DNA passes through. Alternatively,

mut

S fusions

to other

proteins in-

volved in color-generating reactions have been made, and monoclonal antibodies against

mut S are also available. Thus a variety of different methods to exist

to detect

mut

S-DNA

complexes. An attractive feature of

mut S, like carbodiimides, is that it allows the selective

isolation of intact heteroduplexes. The disadvantage shared by both systems is that not all

mismatches are detected. For example,

mut

S

fails to recognize a C–C mismatch, and

some others. The full extent of the advantages and limitations of analysis of mismatches

with

mut S has not yet been described. However, this general kind of approach is attractive

because it mimics a natural biological mechanism, and proteins with properties analogous

to mut

S, but perhaps with even broader mismatch recognition, may well exist in other or-

ganisms.

Two additional methods for detection of heteroduplexes are based on the altered elec-

trophoretic properties of these structures. The simplest of these is direct separation of het-

eroduplexes from homoduplexes of the same length by using specialized gels. A very ef-

fective method uses a modified polyacrylamide

called

MDE, a

term

that

stands

for

mutation detection electrophoresis. This gel has a somewhat hydrophobic character which

alters the mobility of heteroduplexes selectively. Some move faster; most

move slower.

In good cases a single mismatched base in a 900 base pair duplex is sufficient to give an

easily detectable mobility shift. An example of the improved ability of

MDE

compared

with ordinary gel media to resolve heteroplexes from homoduplexes is shown in Figure

13.17. A more complex example of the use of MDE is illustrated in Figure 13.18, where

it is clear that different heteroduplexes within the same basic DNA fragments show differ-

ent mobilities. Thus, in principle, the method offers some promise of revealing, not just

that a heteroduplex is present but additional information about its characteristics. The ap-

peal of this method is that it is very easy to perform, since aside from the special proper-

ties of the gel, the electrophoretic procedures used are quite ordinary. It is also easy to an-

alyze many samples in parallel. However, the full generality of the method has yet to be

proved. For example, it would be good to know how the nature of the mismatch and its

location

within the duplex affect the ability to

detect

it. Already

we know

that deletions

HETERODUPLEX DETECTION

461

Figure 13.19

Denaturing gradient gel electrophoresis (DGGE) detection

of

heteroduplexes. (

a )

Destabilization of a duplex by mismatching. (

b ) Electrophoresis in a temperature or denaturant gra-

dient. ( c ) Use of a GC clamp to prevent strand separation in DGGE.

melts. The

resulting

Y -shaped

structure (or one with a large internal loop) has little or no

electrophoretic mobility, and so the molecule

is

trapped in the

gel

near

or

at

the

site

where it melted (Fig. 13.19

b ). Thus from a single experiment one can determine not only

whether heteroduplexes are present but

also what kinds of mismatches were present,

since most give characteristic and different shifts in

T m .

It is important that the mismatch does not destabilize the duplex so much that the en-

tire structure melts into separate strands. These

would

still

be free to migrate in

the

gel.

To prevent this unwanted effect, a G

C-rich sequence is usually placed at one end of the

duplex to be tested. This is easily

done

by using PCR primers with an extra overhanging

G C-rich sequence. The use of this so-called GC clamp (Fig. 13.19

c ) prevents complete

melting under typical DGGE conditions. A major advantage of DGGE is its generality.

Another advantage is that rather complex samples can be analyzed by running a gel com-

posed of a restriction enzyme digest of a clone and then blotting it with particular probes.

A major disadvantage of DGGE is that specialized apparatus is needed for this method.

A third electrophoretic method we

will

describe

for

detection

of

altered

DNA se-

quences is also based on electrophoresis,

and it takes advantage of the effects of altered

DNA sequence on thermodynamic stability.

However, in detail, this method is actually

quite different from DGGE; it does not involve heteroduplexes. Called single-strand con-

formational polymorphism (SSCP), the method is based on nondenaturing gel elec-

trophoresis

of melted

and rapidly

cooled samples. Under

these

conditions

individual

Sommer, 1995).

A fourth electrophoretic method takes advantage of

the power of automated fluores-

cent DNA sequences. A mutation shows up as an unexpected

peak. The sensitivity of the

only a few copies of the DNA (or RNA,

for some viruses) genome

per

organism. The

number of infected cells (or the number

of organisms free in the

blood

stream or other