Добавил:

fench Опубликованный материал нарушает ваши авторские права? Сообщите нам.

Вуз:

Сумский государственный университет

Предмет:

Генетика

Файл:

Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 7

.pdf

Скачиваний:

Добавлен:

17.08.2013

Размер:

277.57 Кб

Скачать

☆

<<< < Предыдущая 1 23 / 33

228		CYTOGENETICS AND PSEUDOGENETICS
	BOX 7.1 (Continued)
	BOX 7.1 (Continued)
	QUANTITATIVE HIGH-RESOLUTION FISH
	mately linear function of the true distance, out to			about 1.5 Mb (Fig.		7.19	b ). Thus
	these results support the use of a random walk model. The difﬁculty with the available
	results to date is that they do not agree on the scaling. Three studies are summarized
	below:
		INVESTIGATOR	DISTANCES MEASURED			REAL
		Lawrence		1		1 Mb
		Trask		1		0.5 Mb
		Skare		1		1.2 Mb or more
	It is not clear if these considerable differences are due to				differences in the methods
	used to prepare the interphase chromatin or differences in the properties of interphase
	chromatin in different regions of the genome. Note that			the scaling, at worst, is		within
	a factor of three of what was estimated in the text from a very crude model for the
	structure of the 30-nm ﬁlament.

	If this is taken to be a random walk, then the measured			distance	between markers should
	be proportional to the square root of the real distance between them (Fig. 7.17						c;see	also
	Box 7.1).
	An example of the utility of interphase FISH is shown in Figure 7.20. Here interphase
	in situ hybridization was used to estimate the size of a gap that existed in the macrore-
	striction map of a region of human chromosome 4 near the tip of the short arm and now
	known to contain the gene for Huntington’s disease.			The gap was present because no
	clones or probes could be found between markers E4 and A252. In all other segments in
	the region, the accord between distances on the macrorestriction map and distances in-
	ferred from	interphase in situ hybridization	(using a	scaling	of 0.5 Mb per		) is	quite

good. This has allowed the conclusion that the gap in the physical map would have to be small to maintain this consistency.

Figure 7.20 Comparison of a macrorestriction map of the tip of the short arm of human chromosome 4 with interphase FISH results for the same set of probes. (Adapted from Van den Engh et al., 1992.)

CHROMOSOME PAINTING

229

CHROMOSOME

PAINTING

For most chromosomes a dense set of mapped markers now exists. An even larger set of

clones is available and assigned to a particular chromosome but not yet mapped. These

clones can be used to examine the state of the entire chromosome, either in metaphase or

in less condensed states. This practice is called

chromosome

painting.

Metaphase

chro-

mosome painting is a useful tool to see

if a chromosome is intact or

has

rearranged

some way. A novel application of this is

illustrated in Figure 7.21. Here

the

high

degree

of homology between chimp and human DNA sequences was used to examine the rela-

tionship between human chromosome 2 and its equivalent in the chimp. Probes from hu-

man chromosome 2 were used ﬁrst on the human, where they indicated even and exhaus-

tive coverage of the chromosome. Next the same set of probes

was

used

the

chimp.

Here two smaller chimp chromosomes were painted, numbers 12 and 13. Each of these is

acrocentric, while human chromosome 2 is metacentric. These results make it clear that

human chromosome

must have

arisen by

a Robertsonian (centromeric)

fusion

the

two smaller chimp chromosomes.

When interphase chromatin is painted, one can observe the cellular location of particu-

lar segments of DNA. The complexity of interphase chromatin makes it difﬁcult to view

more than small DNA regions simultaneously. This technique is still in

its infancy,

but it

is already clear that it has the potential to provide an enormous amount of information of

how DNA is organized in a functioning nucleus (Chandley et al., 1996, Seong et al.,

1994).

A recently developed variation of chromosome painting shows considerable promise

both in facilitating the search for gene involved in diseases like cancer and in assisting the

development of improved clinical diagnostic tests for chromosomal disorders. In this

technique,

called

comparative genome hybridization

(CGH), total DNA from two sam-

ples to be compared is labeled using different speciﬁcity tags. Actual procedures use nick

translation: For one sample a biotinylated dNTP is used; for the

other

digoxigenin-

labeled dNTP is used (see Chapter 3). These two samples are then allowed to hybridize

simultaneously to

metaphase

chromosome

spread. The results

are

visualized by

two-

Figure 7.21 An example of metaphase chromosome painting, in which a human chromosome 2 probe is hybridized to the full set of human chromosomes, and separately to the full set of chimpanzee chromosomes. Shown are just those resulting chromosomes from both species that show signiﬁcant hybridization.

230 CYTOGENETICS AND PSEUDOGENETICS


Figure 7.22	An example of comparative genome hybridization. Shown is the green to red color
ratio seen along human chromosome 2 after competitive hybridization with DNA		from two	cell
lines. The red cell line is normal human DNA. Three regions of DNA ampliﬁcation are apparent in
the green labeled cell line. The most prominent of these corresponds to the oncogene		N-	myc	known
to be ampliﬁed in the cell line used. (From Kallioniemi et al., 1992.)

color detection using ﬂuorescein-labeled streptavidin or avidin to detect the biotin and rhodamine-labeled antidigoxigenin to detect the digoxigenin. The ratio of the two colors

should	indicate the	relative amounts of the two probes hybridized. This should be the
same if	the initial	probes have equal concentrations everywhere in the genome. However,

if there are regions in one target sample that are ampliﬁed or deleted, the observed color ratio will shift. The results can be dramatic as shown by the example in Figure 7.22. The color ratio shifts provide an indication of the relative amounts of each ampliﬁcation or

deletion,	and they also allow the	locations of	all	such	variations between	two samples to
be mapped in a single experiment.
CHROMOSOME	MICRODISSECTION
Frequently	the search for a gene	has focused	down	to a	small region of a	chromosome.

The immediate task at hand is to obtain additional DNA probes for this region. These are needed to improve the local genetic map and also to assist the construction of physical maps. They may also be useful for screening cDNA libraries if there is any hint of the preferred tissue for expression of the gene of interest. The question is how to focus on a small region of the genome efﬁciently. One approach has been microdissection of that region from metaphase chromosomes and microcloning of the DNA that results. Two approaches to microdissection are shown schematically in Figure 7.23. In one, a ﬁne glass needle is used to scratch out the desired chromosome region and transfer the material to a

site	where it can be collected. In the other		approach, a laser is used			to ablate all of the
genome except for the small chromosome region of interest. In either case, the problem in
early	versions	of this method was to develop efﬁcient		cloning schemes	that could start
from	the very	small amounts of DNA that	could	be collected	by	microdissection.

CHROMOSOME MICRODISSECTION

231

Figure 7.23	Two	examples of chromosome microdissection. (	a ) A ﬁne needle is used to scratch a
chromosome and	transfer a	section of it to another site where it can be further manipulated. (	b ) A
laser is used to destroy the DNA in all but a preselected segment of a chromosome.

Hundreds

microdissection

products would be combined and placed in liquid micro-

drops suspended in oil (Fig. 7.24). Restriction enzymes, vectors, and other components

needed for cloning were delivered

micromanipulators.

The result

was

technical

tour

force

that did deliver clones from the desired region but usually in very small

numbers.

With

the

development

of genome-wide PCR ampliﬁcation methods (Chapter 4),

the

need

for

microcloning

dissected

chromosome

samples

eliminated.

Using

these

methods,

relatively

easy to amplify the dissected

chromosome

material

PCR

until there

enough

DNA

handled

conventional

methods.

Note

that the microdissection method has

several important

potential

uses

beyond

the hunt

for

speciﬁc

disease genes.

Most

DNA

libraries in

any

type

of vector are

biased, and

some regions are under-represented or not represented at all. Microdissection offers an attractive way to compensate for cloning biases, especially when they are severe. The alternate approach, in common use today, is to combine clones from different types of libraries in the hope that biases will compensate for each other. This can be a very effective strategy, but it increases substantially the number of DNA samples that must be handled. Another potential use for microdissection will be to pull clones from a particular individual who may have a region of special interest, such as a suspected rearrangement. This

will not always be an effective strategy, but it may well be necessary in cases where simpler approaches fail to yield a deﬁnitive picture of what has happened.

Figure 7.24 Microcloning of DNA in a tiny droplet containing microdissected chromosomes.

232 CYTOGENETICS AND PSEUDOGENETICS

SOURCES AND ADDITIONAL READINGS

Boyle, A. L., Feltquite, D. M., Dracopoli, N. C., Housman, D. E., and Ward, D. C. 1992. Rapid

physical mapping of cloned DNA on banded mouse chromosomes by ﬂuorescence

in situ hy-

bridization.

Genomics

12: 106–115.

Burmeister, M., Kim, S., Price, E. R., de Lange, T., Tantravahi, U., Meyers, R. M., and Cox, D.

1991. A map of the distal region of the long arm of human chromosome 21 constructed by radi-

ation hybrid mapping and pulsed ﬁeld gel electrophoresis.

Genomics

9: 19–30.

Chandley, A. C., Speed, R. M., and Leitch, A. R. 1996. Different distributions of homologous chro-

mosomes

adult human Sertoli cells and in

lymphocytes signify nuclear differentiation.

Journal of Cell Science

109: 773–776.

Cherif, D., Julier, C., Delattre, O., Derré, J., Lathrop, G. M., and Berger, R. 1990. Simultaneous lo-

calization of cosmids and chromosome R-banding by ﬂuorescence microscopy: Application to

regional mapping of human chromosome 11.

Proceedings of

the National Academy

of Sciences

USA

87: 6639–6643.

Cox, D. R., Burmeister, M., Price, E. R., Kim, S., and Meyers, R. 1990. Radiation hybrid mapping:

A somatic cell genetic method for constructing high-resolution maps of mammalian cell chro-

mosomes.

Science

250: 245–251.

Ellis, N., and Goodfellow, P. N. 1989. The mammalian pseudoautosomal region.

Trends in Genetics

5: 406–410.

Heiskanen M., Kallioniemi, O., and Palotie, A. 1996. Fiber-FISH: Experiences and a reﬁned proto-

col.

Genetic Analysis (Biomolecular Engineering)

12: 179–184.

Jeffreys, A. J., Neumann, R., and Wilson, V. 1990. Repeat unit sequence variation in minisatellites:

A novel source of DNA polymorphism for studying variation and mutation by single molecule

analysis.

Cell

60: 473–485.

Kallioniemi, A., Kallioniemi, O. P., Sudar, D., Rutovitz, D., Gray, J. W., Waldman,

F., and Pinkel,

D. 1992. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.

Science

258: 818–821.

Lawrence, J., Singer, R. H., and McNeil, J. A. 1990. Interphase and metaphase resolution of differ-

ent distances within the human dystrophin gene.

Science

249: 928–932.

Li, H., Cui, A., and Arnheim, N. 1990. Direct electrophoresis detection of the allelic state of single

DNA molecules in human sperm by using the polymerase chain reaction.

Proceedings of the

National Academy of Sciences USA

87: 4580–4584.

Li, H., Gyllensten, U. B., Cui, X., Saiki, R. K., Erlich, H. A., and Arnheim, N. 1988. Ampliﬁcation

and analysis of DNA sequences in single human sperm and diploid cells.

Nature

335: 414–417.

Lichter, P., Ledbetter, S. A., Ledbetter, D. H., and Ward, D. C. 1990. Fluorescence in situ hybridiza-

tion

with

Alu

and

L1 polymerase chain

reaction probes

for rapid

characterization

of human

chromosomes

in hybrid

cell

lines.

Proceedings

of the National Academy of

Sciences USA

87:

6634–6638.

Lichter, P., Tang, C. C., Call, K., Hermanson, G., Evans, G. A., Housman, D., and Ward, D. C.

1990. High resolution mapping of human chromosome 11 by in situ hybridization with cosmid

clones.

Science

247: 64–69.

McCarthy, L. C. 1996. Whole genome radiation hybrid mapping.

Trends in

Genetics

12: 491–493.

Nilsson, M., Krejci, K., Koch, J., Kwiatkowski, M., Gustavsson, P., and Landegren, U. 1997.

Padlock probes reveal single nucleotide differences, parent of origin and in situ distribution of

centromeric sequences in human chromosomes 13 and 21.

Nature Genetics

16: 252–255.

Ruano, G., Kidd, K. K., and Stephens, J. C. 1990. Haplotype of multiple polymorphisms resolved

by enzymatic ampliﬁcation of single DNA molecules.

Proceedings

of the National Academy

Sciences USA

87: 6296–6300.

				SOURCES AND ADDITIONAL READINGS			233
Seong, D. C., Song, M. Y., Henske, E. P., Zimmerman, S. O., Champlin, R. E., Deisseroth, A. B.,
and Siciliano, M. J. 1994. Analysis of interphase cells for the Philadelphia translocation using
painting probe made by Inter-			Alu	-polymerase chain reaction from a radiation hybrid.		Blood	83:
2268–2273.
Silva, A. J., and White, R. 1988. Inheritance of allelic blueprints for methylation patterns.						Cell	54:
145–152.
Stewart, E. A., McKusick, K. B., Aggarwal, A., et al. 1997. An STS-based radiation hybrid map of
the human genome.		Genome Research		7: 422–433.
Strong, S. J., Ohta, Y., Litman, G. W., and Amemiya, C. T. 1997. Marked improvement of PAC and
BAC cloning is achieved using electroelution of pulsed-ﬁeld gel-separated partial digests of ge-
nomic DNA.	Nucleic Acids Research			25: 3959–3961.
Van de Engh, G., Sachs, R., and Trask, B. J. 1992. Estimating genomic distance from DNA se-
quence location in cell nuclei by a random walk model.				Science	257: 1410–1412.
Wang, D., and Smith, C. L. 1994. Large-scale structure conservation along the entire long arm of
human chromosome 21.		Genomics		20: 441–451.
Wu, B-L., Milunsky, A., Nelson, D., Schmeckpeper. B, Porta, G., Schlessinger, D., and Skare, J.
1993. High-resolution mapping of probes near the X-linked lymphoproliferative disease (XLP)
locus. Genomics		17: 163 – 170.
Yu, J., Hartz, J., Xu, Y., Gemmill, R. M., Korenberg, J. R., Patterson, D., and Kao, F.-T. 1992.
Isolation, characterization, and regional mapping of microclones from a human chromosome 21
microdissection library.		American Journal of Human Genetics			51: 263–272.

<<< < Предыдущая 1 23 / 33

Соседние файлы в папке genomics1-10

#
17.08.2013435.19 Кб502.pdf
#
17.08.2013343.56 Кб463.pdf
#
17.08.2013296.13 Кб464.pdf
#
17.08.2013326.85 Кб465.pdf
#
17.08.2013406.31 Кб456.pdf
#
17.08.2013277.57 Кб467.pdf
#
17.08.2013634.83 Кб478.pdf
#
17.08.2013475.69 Кб469.pdf
#
17.08.2013192.47 Кб48booktext[1].pdf