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

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490

SEQUENCE-SPECIFIC MANIPULATION OF DNA

which avoid the problem of multiple

stereoisomers. Among

the

compounds

that

have

been made and tested are polypeptide nucleic acids, in which a peptide backbone

is used

to replace the phosphodiester backbone, and polyamide

nucleic

acids

(PNAs).

The

schematic structure of one example of this latter class of compounds is shown in Figure

14.23.

An amusing series of accidents clouded the ﬁrst attempt to characterize

the interac-

tions of PNAs with duplex DNA. The actual compound used was R1-T10-R2. The nota-

tion T10 means that 10 thymine bases and backbone units were present. R1 was chosen to

contain a positive charge for extra stability of interaction of the short PNA with DNA. R2

contained an intercalating acridine, also present to enhance binding stability, and a p-ni-

trobenzoylamide group. This latter promotes radical-induced cleavage of the DNA upon

irradiation with near-UV (30 nm) light. This PNA was designed with the expectation that

it would bind to a dA-dT stretch in a duplex and form a triple strand. What actually hap-

pened was more complex. A number of

different

chemical

and

enzymatic probes

were

used to examine the resulting PNA-DNA complex. All were consistent with the idea that

the PNA had displaced the dT-containing strand of the natural duplex and formed a more

stable duplex with the uncovered dA-stretch. A number of the results that led to this con-

clusion are shown in Figure 14.24

a . The prospects raised by this outcome were extremely

exciting because a duplex strand

displacement

mechanism

would

applicable

any

DNA sequence, not just to the more limited set capable of forming triplexes.

Further investigations of the properties of the PNA-DNA complex reveal an additional

level of complication. It turns out that the complex contains not just one stoichiometric

equivalent of PNA, but two instead. The result, as shown schematically in Figure 14.24

b ,

is a complex in which displacement of one of the strands of the original DNA duplex has

occurred, but the displaced strand is

captured

as a triplex

with two PNAs. Thus

this

sort

of reaction will be limited to sequences with triple-strand forming capabilities. Perhaps

other

backbone analogues will be

found that work by displacing

strands and capturing

Figure 14.23 Chemical structures of the normal DNA backbone and polyamide nucleic acid (PNA) analogs. Adapted from Nielson et al. (1991).

USE OF BACKBONE ANALOGUES IN SEQUENCE-SPECIFIC DNA MANIPULATION

491

Figure 14.24	PNA binding to DNA.	(a) Chemical evidence for the displacement of a (dT) 10 se-
quence by a corresponding PNA derivative.		(b) The structure of the complex actually formed.
Adapted from Nielson et al. (1991).

them as duplexes. An alternative scheme for in vitro work would be the use of recA pro- tein-coated single strands. However, this is most unlikely to be useful in vivo.

In the past few years, a considerable amount of work has been done to explore the properties of PNAs and their potential usefulness in DNA analysis or clinical diagnostics.

A few of these ﬁndings are brieﬂy summarized here. The stability of PNA-DNA or DNA-

PNA duplexes is essentially salt-independent (Wittung et al., 1994). Thus low salt can be used in hybridization procedures such as SBH to supress the interference caused by stable secondary structures in the target. PNAs are capable of forming sequence-speciﬁc duplexes that mimic the properties of double-stranded DNA except that the complexes are completely uncharged. Because there is no chirality in the PNA backbone, the duplexes

are optically inactive; they have no preferred helical sense. However, attachment of a single chiral residue such as an amino acid at the end of the PNA strand leads to the forma-

tion of a helical duplex (Wittung et al., 1995). The ability of PNAs to bind tightly to speciﬁc homopurine, homopyrimidine duplexes leads to an effective form of Achilles’s heel

cleavage (Veselkov et al., 1996). Triplets that are located near restriction enzyme cleavage sites block these sites from recognition by the conjugate methylase. After removal of

the triplex, the restriction nuclease will now cleave only at the sites that were previously

492 SEQUENCE-SPECIFIC MANIPULATION OF DNA

protected, as in Figure 14.11 b . The PNA-mediated protection appears to be quite efﬁcient. A ﬁnal novel use of PNAs is for hybridization prior to gel electrophoresis (Perry-

O’Keefe et al., 1996). Since PNA is uncharged, it can be used to label ssDNA without interfering with subsequent high-resolution electrophoretic fractionations.

SEQUENCE-SPECIFIC CLONING PROCEDURES
Instead	of physical isolation of particular DNA	sequences, cloning or PCR procedures
can be	used to purify a desired component from a	complex mixture. Direct PCR is very

powerful if some aspect of the target DNA is known at the sequence level (Chapter 4). Where this is not the case, less direct methods must be used. Here several procedures will be described for speciﬁc cloning based indirect information about the desired DNA se-

quences to be puriﬁed. Several PCR procedures that take advantage of the	possession
of only a limited amount of DNA sequence information will be described later	in the
chapter.

Subtractive cloning is a powerful procedure that has played an important role in the search for genes. It can be carried out at the level of the full genome with much difﬁculty, or at the level of cDNAs with much greater ease. In subtractive cloning the goal is to isolate components of a complex DNA sample that are missing in a similar, but not identical,

sample. One strategy for doing this, which illustrates the general principles, is shown in Figure 14.25. In this case, which is drawn from the search for the gene for Duchenne muscular dystrophy (DMD), two cell lines were available. One had a small deletion in the region of the X chromosome believed to contain the gene responsible for the disease. This deletion was actually found in a patient who displayed other inherited diseases in addition

to DMD (the utility of such samples was discussed in Chapter 13). The objective of the

Figure 14.25 Differential cloning scheme originally used to obtain clones corresponding to the region of the genome deleted in Duchenne muscular dystrophy.

					SEQUENCE-SPECIFIC CLONING PROCEDURES			493
differential cloning was to ﬁnd DNA probes that derived from the region that was deleted
in this patient, since these would be candidate materials for the DMD gene itself.
	A small amount of DNA from a normal individual was used as the target. This was cut
with a restriction enzyme to give DNA fragments with cloneable ends. A large excess of
DNA from the patient with the small deletion was prepared and cut into longer fragments
than the target sample. This was done with an enzyme that would not give cloneable ends
in the vector ultimately used. The two samples were melted and mixed together to co-an-
neal. Because the normal DNA was limiting, the DNA from the sample with the deletion
acted as a driver. It rapidly formed duplexes with itself, and with corresponding fragments
of the normal DNA. In contrast, DNA from the region of the deletion was present at very
low concentrations in the mixture, and it renatured very slowly. Once renatured, however,
the resulting duplexes had cloneable ends, unlike all of the rest of the DNA fragments in
the	sample. The mixture was then ligated into a vector				and transformed into a suitable			E.
coli	host. The resulting clones were, indeed, highly enriched for DNA from the desired
deletion region.
	The major difﬁculty inherent in the scheme shown in Figure 14.25, is that the desired
DNA	fragments are	at	low concentration and	form duplex very slowly		and inefﬁciently.
In fact, to achieve an acceptable yield of clones, the renaturation had to be carried out in a
phenol-water emulsion, which raises the effective DNA					concentration	markedly. This	is
not an easily managed or popular approach. More recent analogs of subtractive genomic
cloning have been described that look powerful, and they should be more easy to adopt to
a broad variety		of	problems (see Box	14.1).	The potential	power of such	schemes is

shown by the mathematical analysis of the kinetics of differential cloning in Box 14.2.

BOX 14.1

NEWER SCHEMES FOR DIFFERENTIAL GENOMIC DNA CLONING

Three schemes for cloning just the differences between two DNA samples will be de-

scribed. The ﬁrst two	were	designed to clone DNA corresponding to a region deleted
in one available source	but	not in another. These schemes are similar to that described

in Figure 14.25 except that they ﬁrst use biotinylated driver DNA to facilitate the separation of target molecules from undesired contaminants, and then they use PCR to amplify the small amount of target molecules that remain uncaptured. In one scheme, de-

veloped by Straus and Ausubel	(1990; Fig. 14.26), an excess of biotinylated driver
DNA is used to capture and remove most of the target DNA by repeated cycles of hy-
bridization and afﬁnity puriﬁcation with streptavidin-coated beads. Then the remaining
desired target DNA is ampliﬁed	and subsequently cloned by ligation of appropriate
PCR adapters.

In a related scheme, developed by Eugene Sverdlov and co-workers, it is the target DNA that is biotinylated by ﬁlling in the ends of restriction fragments with dpppN de-

rivatives (Fig. 14.27; Wieland et al., 1990). This target is then provided with PCR adapters by ligation. Excess driver DNA is used to deplete most of the target by cycles

of hybridization and hydroxylapatite chromatography to remove any DNA duplexes formed. After several such cycles, streptavidin afﬁnity chromatography is used to capture any biotinylated target remaining. The target molecules are then ampliﬁed by

(continued)

494 SEQUENCE-SPECIFIC MANIPULATION OF DNA

BOX 14.1

(Continued)

Figure 14.26 Differential cloning scheme based on repeated cycles of hybridization and bi- otin-afﬁnity capture followed by PCR ampliﬁcation. Adapted from Straus and Ausubel (1990).

Figure 14.27 Differential cloning scheme based on repeated cycles of hybridization and hydroxyl apatite chromatography followed by biotin afﬁnity capture and PCR ampliﬁcation. Based

on a method described by Wieland (1990).

(continued)

SEQUENCE-SPECIFIC CLONING PROCEDURE

495

BOX 14.1

(Continued)

Figure 14.28 A method for cloning the differences between two genomes (RDA). Adapted from Lisityn et al. (1993).

primers complementary to the adapter sequences and cloned. Both of these methods appear to be quite satisfactory, and both can be enhanced, if necessary, by repeating the steps involved.

Recently a scheme has been described by the Lisityn et al. (1994) for cloning polymorphic restriction fragments. This scheme is illustrated in Figure 14.28. It has been

called representation	difference	analysis	(RDA).	First, the complexity of both target
and driver genomes is	reduced by	PCR to	allow more	effective subsequent hybridiza-

tions (Chapter 4). This is done by ligating on adapters and removing them after the PCR ampliﬁcation. Then, as shown in Figure 14.28, the target is provided with new PCR adapters by ligation. Target is mixed with excess driver, melted, and reannealed.

The ends of the duplexes formed are ﬁlled in with DNA polymerase. PCR is now used

to amplify the entire reaction mixture. The key point is that target duplexes will show exponential ampliﬁcation because they contain two adapters. Heteroduplexes will show only linear ampliﬁcation, while driver DNA will not be ampliﬁed at all. Any sin- gle-stranded molecules remaining are destroyed by treatment with mung bean nucle-

ase, a single-strand speciﬁc enzyme similar to S1. Then the cycles of hybridization and ampliﬁcation are repeated.

496	SEQUENCE-SPECIFIC MANIPULATION OF DNA

BOX	14.2
SUBTRACTIVE HYBRIDIZATION
The purpose of subtractive hybridization is to purify a target DNA strand, symbolized
by	T , from other DNA, called tracer DNA, symbolized by	S . This is accomplished by
the use of driver DNA strands ﬂanked by different primers, symbolized by		D . The pro-
cedure is illustrated schematically below. In general, genomic or cDNA samples would
be digested to completion with a restriction nuclease and ligated to splints to prepare
sequences for subsequent PCR ampliﬁcation.

The mathematics behind this procedure is based on an equation developed in Chapter

3 to describe the kinetics of double-stranded DNA formation. If we call the initial con-

centration of single-stranded DNA segments is

c 0 ,

then the fraction of DNA that has

formed double-stranded segments,

, is given by the equation

2 0

1 k

2 0

where

t is the time and

k 2is a constant for that particular sequence of DNA. Using this

equation, we can determine the concentration of double-stranded segments by multi-

plying the initial concentration:

f c

2 0

(continued)

SEQUENCE-SPECIFIC CLONING PROCEDURE

497

BOX 14.2

(Continued)

First Round of Subtraction

Consider two DNA samples. The ﬁrst contains

S-

and

T -type DNA; the second con-

tains only

S -type DNA. The sample containing only

S -type DNA, however, is ﬂanked

by different primers, and will be designated by

D . When

these

two samples

are dena-

tured and mixed, they will form double-stranded segments. The concentrations of vari-

ous species can be determined by the equations above.

Since the

T -type single strand will bind only with other

T -type single strands, we

can ignore the presence of

S - and

-type strands in calculating the concentration of the

double-stranded

duplexesT T

formed. If

c T

is the

initial concentration

T single

strands, the concentration of

double-strandedT T

segments, is

k c

)

round

2 T

1 k c

first

2 T

calculate

the concentration

of the double-stranded

tracer

assume that

S ,

the concentration

insigniﬁcant

compared to the

concentration

. Double-

strand

formation

S , S

, and willD occurD

indiscriminantly. Thus

we can

ﬁrst

compute

the kinetics

formationD andD

then

extract

the

amount

by multi-

S S

plying by the mole fraction of

denoted byS S ,

XS S :

XS S

Where ciss 1the initial concentration of S-type strands and tion of D -type strands. Therefore

c s21

c 2

D 1

is the initial concentrac-D 1

k c

2 t

)

round

)

round

2 D 1

s 1

2 s 1

1 k

first

D ds

first

2 D

)

round

and ( c

S ds

)

are the concentrations of double-stranded

and

S S

first

first round

structures.

The next step in the subtraction protocol shown above is to amplify the strands by

PCR. Since only

the

andT

strandsS

haveS matching

primers,

only

these

strands

will be ampliﬁed exponentially. This

results

in the

effective

removal of

and

D D

strands, reducing the amount of

strand

contamination,

while

increasing

the

concen-

tration of

strands.

The ratio of concentration of

versus theT concentrationT

can now be cal-

S S

culated. This ratio is the enrichment resulting from the ﬁrst subtraction step:

first

1 k c

T ds

E first

round

2 T

2 D

S ds

1 k

c 2t

1 k

2 T

2 S 1

S 1

2 T

Since the initial round of subtraction is performed with samples

directly

from

the

genome,

the

concentration

strands is

the

same

as the concentration of

strands.

(continued)

498 SEQUENCE-SPECIFIC MANIPULATION OF DNA

BOX 14.2

(Continued)

This means that the ratio

2 2

t, the en-

is equalc

/toc

one for the ﬁrst round. So, for large

richment ratio is

S 1

E first

T ds

c D

round

S ds

first

round

c T

indicating that by the end of the ﬁrst round, the ratio between

and

will be

as large

as the initial ratio of

and

DNAs

used. Simply

using a

much higher

concentra-

tion of

strands than

strands, the presence of

S -type strands can be signiﬁcantly re-

duced.

Second Round of Subtraction

In following the procedure illustrated above for a second round of subtraction, it is im-

portant to note that the initial concentrations of

S , T , and

for

the

second round are

related to

their concentrations

at the end of the ﬁrst round. The initial concentration

T strands for the second round can be assumed to be the same as the initial concentra-

tion

strands

for the

ﬁrst round. The concentration of

strands,

however,

will

less than that of the ﬁrst round and will be denoted by

. Since the PCR ampliﬁcation

in the ﬁrst round should not discriminate between

and

s 2

T , we can assume that

c s 2

c S ds

c T

first round

c D

The

concentration of

D strands

for the second round

much

greater

than

either

c S 2

c T . This value will be called

c D 2.

the

second

round

proceed

exactly

the

Calculations for the annealing kinetics in

same way as in the ﬁrst, except that we now use the values

c T

, c s 2, and

c D 2:

)

2 T

second round

2 D

)

2 s 2

second round

2 D

PCR ampliﬁcation at this point replenishes the amount of

T -type strands, while effec-

tively removing some of the

S -type strands. The

ratio between the concentrations of

and

can

be calculated,

as in

the

ﬁrst round,

from the

initial concentrations

for

the

second round:

k c

1 k

1 k c

T ds

2 T

2 D

second round

k c

1 k c

S ds

2 T

2 S 2

S 2

2 T

(continued)

IDENTIFICATION OR CLONING OF SEQUENCES BASED ON EXPRESSION LEVEL		499
BOX 14.2	(Continued)

For large t, the ﬁnal ratio of concentrations in the second round can be simpliﬁed to

T ds

c 2

second round

D 2

E first2 round

S ds

c 2

second round

S 2

Since c D is2 chosen

arbitrarily,

if we use

the

value

again,

the ratio

ofc D the1

ﬁnal con-

centration of the second round simpliﬁes even further:

E second round

E first2 round

c D

E first3 round

c T

This is a remarkable result which shows that multiple subtraction protocols have a puriﬁcation power that increases unexpectedly (adapted from notes provided by Eugene Sverdlov, as formulated by Ron Yaar).

c D 2 c T

If the starting DNA samples are genomic restriction fragments, the resulting ampliﬁed
products eventually recovered		will be those fragments in the subset of originally	ampli-
ﬁed material that had one restriction site that differed in the driver DNA. Such polymor-
phisms identiﬁed have been called polymorphic ampliﬁable restriction endonuclease frag-
ments (PARFs). There are estimated to be around 1000 such			Bam	H I fragment
differences between any two human genomes. Thus PARFs offer a potentially very pow-
erful way to obtain useful genetic probes near preselected regions if DNA from appropri-
ate individuals is available. For example, suppose that one has a population of individuals
heterozygous for a dominant trait of interest. Subtraction of the DNAs of subsets of this
population with DNAs from related individuals who lacked the trait should offer a rea-
sonable chance of producing clones that contain polymorphisms linked to the trait or even
responsible for the trait. A number of interesting variations on the original differential
cloning scheme have been described (Yokata and Oishi, 1996; Rosenberg et al., 1995;
Inoue et al., 1996). It remains to be seen how well such potentially very exciting new
strategies actually perform in practice.
IDENTIFICATION OR CLONING OF SEQUENCES BASED ON
DIFFERENCES IN	EXPRESSION	LEVEL
Once DNA sequences of potential interest have been identiﬁed, a frequent next step in
understanding their function is to determine when and where in the organism they are ex-
pressed. For a simple sequence of interest, a suitable analytical method is the			Northern
blot. Here mRNAs from tissues or other samples of interest are fractionated by length us-
ing gel electrophoresis, transferred to a membrane and hybridized with a probe speciﬁc to
the gene of interest. This is called a Northern blot, and it is a widely used procedure for
accessing the expression level of individual genes. An alternative method is quantitative
PCR (qRT-PCR). qRT-PCR requires much lower sample amounts but is difﬁcult to stan-
dardize because of the intrinsically variable characteristics of PCR. Some protocols add a
standard amount of	target mimic to the PCR reaction. Since the mimic is usually		shorter

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