Note: Descriptions are shown in the official language in which they were submitted.
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;.. WO93/22463 PCI~US93/04330
METHODS FQR HIGH RESOLUrlON GENE MAP~ING
Back~ of the Invention
The human ~enome consists of about 3 billion b~se pairs of i~NA carried on 46
ohromosornes. This ~enetic blueprint encodes the information required for the ~rowth,
0 cilfferentlation, maintenance and prop~r functionln~ of human cells. To aid inIcientifyin~ ~enes assoclated wlth disease, there has b~en ~reot interest in mappin~
the human 0enome. Indeed, in creating the Human Genome Pro3ect, the United
States ~ovsrnment has announced the elucidation of the human genome as a
r~ lonal objective.
The two maln types of hurnan ~enome maps are ~enetic linka~e and
physlcal, Genetlc linka~c maps are ~eneratsd malnly by studyin~ families and
measurin~ the frequancy with which two different traits are inher~ted toç~ether, or
l~nked. Physical maps. on the other hand, are derived from measuraments made on
the DNA rnolecules that fomn the human ~enome.
Physical maps ~an be created baseci on inform~tion provided by restriction
; fra~ament polymorphisms or a collection ot ordered clones of ~enomic fra~ments (e.g.
~; cosmlds). Maps based on ordered clones are especially useful, since each fragment
is avallaijle as a clone that can be propa~ated and ciistributed. These clones can
serve as a startin~ point for ~ene isolation, functional analyses anci for the
2s cietermination of nueleotide sequences~ Preparin~ an ordered-clone co~lectionInvolves clonin~ DNA fra~ments, determinin~ their order In the genome and
prcpa~atin0 the fra~ments in pure form to make them wldely ava~lable for
subsequent analysis. Because of the limited resolutlon currently available, the most
difficuit aspect involves determlnin~ the order of DNA fra~ments~
1 I`rt situ hybri~i~ation clnalysis h~s been us~ul for ~ehome mappin~, because!it
~;~ allows direct detection of the location at which a DNA fra~ment maps on a particuiar
chromosome.~ Howevar, the resolution for iocalizin~ probes to chromosomal bands in
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metaphas& -hromosomes is only several me~abases (Lichter, P., et. al., et. al., Science
247~ 9 ~ Lawrence, J.B., et. al., Science 249:64-69 (1990)). To achieve higher
rasolution, ~iSH has been ~pplied to cell tar~ets collected in interphase (Lawrence, J.B.
et. al., Cell 52:51-61 (lQ88)), At interphase, as opposed to at metaphase, chromatin
within the cell nucleus exists in an uncondensed form. Probes separated by distances
from c100 K up to at least lMb have been ordered on the X chromosome in somatic
Interphase nuclel (Lawrence, J.B., et. al., Science 24~ 9 (1990): Trask, B.J., et. al.,
Am. J. Hum. Genet. 48:1-15 (1991)). Usin~ male interphase pronuclei generated by __ _
~ amete fusion, probes separated by less than ~Okb have been rssolved. (Brandriff, B.,
0 et. al., Genc)rnlcs 10:7~82 (1991)). However, this technique is time consumin~, requires
a hi~h level of sklll ancl requires materials that are not readily available to most
researchers,
Niumerous probes are ~enerated in efforts to ident fy ~ene loci. A need exists
for a simple and effective hi~h resolution method for determinin~ the relative order of
these prol~s and to determinè their pOsnion relative to known markers.
Sum ary of the Invention
In ~eneral, the inventian relates to rnethods for oraierin~ at least two discrete
20 DNA sequences on a chromosome. in one embodiment, the inventlon fe~tures a)
contactin~ a eukaryotic cell containin~ a chromosome of interest with an agent that
effects dscondensation: b) preparin~ the cell containin~ decondensed chromatin for
hybridization: and c) hybridizin~ the cell produced in step b) w~h at ieast two i3NA
probes, each probe bein~ complernent~ry to at least or e discrete DNA sequence
25 present on the chromosome of interest: and d) detectlng the presence and relative
order of the probes as an indicatlon of the relative and absolute locations of the DNA
sequences on the chrornosome. By decondensin~ interphase chromatin according
to the rnethod of the invention, hi0h resolution interphase rnappin~ can be carried
out. In effect, fra~ments containin~ DNA sequences that abut one another or even30 overlap can b- resolved. In addition, because the methods disclosed her0in render
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the DNA more accessible to hybridization, results are more readily obtained.
In another embodiment, the invention features orderin~ at least two discrete
DNA sequences on a fra~ment of DNA that has been extencied, for example by
physlcal means, chernic~l means or a combination of both. A preferred physical
s rneans for extendln~ ONA is by ~entle smearing. A preferred chemical means forobtainln0 extended DNA from a eukaryotic ~enome is by contacting the chromatin
with a solution that removes hlstones~ Once extended, the DNA can be prepared for
hybridlzation and hybridlzed with at least two DNA probes, each probe being
complementary to at least one discrete DNA sequence present on the chromosome
10 of IntQrest. Once hybridized, the presence and relative order of the probes can be
determlned as an indication of the relative and absolute locations of the DNA
sequences on the fra~ment of DNA. By extendin~ DNA accordin~ to the method of
the invention, mappin~ can be carried out at an even hi~her resolution.
15 Detailed Description af the Invention
The subiect invention is baseci on the discovety that chrornosornes which are
^relaxed~ or DNA whlch is ~extendeci~ can be analyzed usin~ a hybridization assay to
detcrminé the order in which a set of DNA sequences ~i.e. at least two) map on a20 partlcular chrornosome or fr~ment of DNA. The Invention, therefore, features two
convenient methods for orcierin~ a set of dlscrete DNA sequences at a hi~h resolution.
The ordering of discrete DNA sequences is useful for example for physical mapping.
As ussd hersln, the term ~orderin~ rneans establishin~ the linear relationship of
dlscrete DNA seciuences relat'lve to one another and/or relative to a known marker on
2 5 a chromosome or portion of a ~hromosome.
In one aspQct of the subJect invention, a cell is contacted with an agent that
effects decondensc~tion,~ prepared for hybrldkatlon and analyzed usin~ at least two
.
DNA probes, each probe bein~ complementary to at least one discre~e DNA
: sequence prssent on a chromosome of Interest. Preferably the cells are in interphase,
30 so tha~the chromatin is,~already in a somewhat extended~,form. The gro~th of cells in
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culture can be synchronized using known methods. By using synchronized cells, a
hi~her proportion of cells In the interphase stage of the cell cycle can be obtained.
Accordin~ to the method of the subject invention, a cell is first contacted
with an a~;7ent that effects decondensation~ For example, an agent that effects
s deconciensation can be a chemical which inhibits proteins that deacetylate histones
(i.e. the set of proteins that interact with DNA to form chromatin). When histones are
hyperacetylated, the chromc~in becomes l~ss condensed or relaxed. A salt of a short
chaln fat~y acid (such as sodium butyrate, sodium propionate and sodium valer~te)
when added to cells appears to relax chromatin, c3pparsntly by inhlbitin~ the proteins
0 that deacet~late histones.
Cells containin~ relaxed chromatin ~ra then prepared for in situ hybridization
analysis usln~ methods, which clre well-known in the art. In general such methods
involve deposHlnç~ celis on a solid substrate, fixin~, dryin~ and den~urin~ them to yield
sln~le stranded DNA that Is therefore available for hybridization. Cells can be
15 deposited on a substrate such as glass, plastic or nHrocellulose. A glass microscope
sllde Is preferred, because It can be readily manipulated and viewed under a
mlcroscope. The substrate c~n b~ pretreated with a ~cell adherent" which irnproves
the llke!lhooci th~t a cell settlin~ onto the surface remalns attached during subsequent
manipulc~tlons. A preterr~d cell adherent is 3-Aminopropyitriethoxysilane. Treatment
20 with this adherent results In 'silanizeci~ su~strcnes. C)ther adherents include poly-L Iysine
and mussel adhesin. Pretreatment o~ solid sa~bstrates can be accomplished usin~ any
method th~t ensures that a cell adherent is d~posited (e.~. submersian, trclnsferring
usin~ a dropper, etç.). Pretreated solid substrates can be stored in a dust freeenvironment at room temperature.
Cells on a substrate can then be processed through a fixotion protocol to
pres~rve the nuclei/ chromosorne in a morpholo~ically stable state so that nucleic
acids are retained throu~h the ri~orous conditiorls present during in situ i ybridi~ation.
Approprlate fixatives are weil-known in the art and include, for example, 4%
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paraformaldehyde or ~lutoraldehyde in phosphate buffersd solution (Pi3S) containing
5mM MgCI2, a fixative containin~ 3 parts ethanol and 1 part acetic acid, Carnoy's
flxative, 1% osmlum tetraoxide, Bouin's ~ixative, Zenker's fixative.
Prapared cells containin~ relaxed chromatin can then be hybridized with
5 appropriat0 probes and detected using methods which are wel~known in the art, e.g.
hybrldizatlon and det~ction can be performed basically as described by Lichter et. al.
(Hum. Genet. 80:224-234 ~1988)~.
For use In the subJect invention, deoxyribonucleio acid (DNA) probes, labelled
with a detectable marker can be preparad from sin~le-stranded ~NA molecules or
10 fra~ments thereof accordin~ to procedures which are well known in the art. Such
techniques include Incorporation of radioactive l~bels, direct ~ttachment of
fluorochromes or enzymes, and various chemical modiflcations of the nucleic acidfra~msnts that render them detectable immunochemic~lly or by other affinity
reactions. A preferred method of labellin~ Is by nick translation using a haptenated
15 nucleotide triphosphate (e.~. blotin laballed dUTP) or by random primer e~tension
(Felnbery & Vo~elstein, Anal. Biochern. 137:2~ 267 (1984~ (e.g. multiprime DNA
labelin~ system (Amersham~ substitutin~ dTTP with Bi~1 1-dUTP. (Langer, P.R., et. al.,
Proc. Natl. Acad. Scl. USA, 78:S633-37 (1981); Briyati, D.J., et. al., Virolo~y, 126:32 50
(1983))~
For DNA sequenc~ ordering, ~ach of the probes should ~ ciistinctly labelled.
Three sets of dlstln~ulshable fluorophores, emlttin~ In the ~reen (e,g fluoroscein), in the
red (e.g. rhodamine or Texas Red), and in the blue (e.~. AMCA or Cascade Blue) are
typically useci for fluorescent In situ hybridization (FISH). Therefore using standard
procedures, at leost three probes can be used in combination for DNA sequence
2s orderin~ More than three probes can be dete~ted simuitaneously usin~ the methods
of Reid et~ al~, (Proc. Natl. Acad. Sci. USA, in press, 1992~. DNA sequence ordering not
only can be açcomplished by datermining the position of probes relative to one
another, but orderln~ ean also make use o~ known chromosome markers (e.g.
centromares and t~lomeres).
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In another embodiment, the invention fec~t~Jres orderin~ at least two discrete
DNA sequences on a fra~ment of DNA that has iieen extended. Such DNA can be
obtalned, for exarnple frorn a eukaryotie or a prokclryotic chromosome, atthoughhlstones must first be removed from DNA obtained from a eukaryotic chromosome.
5 Histones can be removed chemically usin~ a histone remov~n~ solution ~e.g. see P~uison and Laemmli Cell 12:817 828 (1977)).
DNA ~ree in solution (e.~. DNA extracted from a prokaryote or DNA extracted
from a eukaryotic chromosome and treated to remove proteins) can be extended by
physical means. A preferred physical rneans for extendin~ DNA is by gentle smearinç~
10 or pullin~ of the DNA, e.~. usin~ a pippette. Once extended, DNA can be prepared
for hybridlzatlon and hybridized with appropriate probes as descrl~ed above to
determine the rel~tive ~nd absolute locations of the DNA sequences on the fragment
of DNA.
The present example will now be further illustrated by the following examples,
whlch are not intended to be limitin~ in any way.
Example 1: Preparation of Nuclel Containin~ Decondensed Chromatin
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1. Short term Iymphocyte cuitures were establisheci usinçj standard
~ 2 0 protocols and incujated for 72 hrs c~t 37C.
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2. ~iium b~yrate was added to cultures to a concentrcrtion of 7mM
(from a 500mM stock) and incubated an addltional ~6 hrs at
37C.
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3. Celis were resuspendeù In BT b~fer (20mM sodium butyrate, lmM
; Tris, 25mM KCI, 0.9mM M0C12 and 0.9mM CaC12 at pH 7.6).
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4. Cells were resuspend0d in BT/PMSF.(BT buffer plus 0.5mM
i30 ~ phenylmethytsu~fony!fluoride(PMSF)).
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~ 5. Celis were resus;~ended in i3T buffer, then several drops af
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Carnoy's fix (3 parts methanol: 1 part acetic acld) was acided
;; ~ ~ to the celi suspenslon and gently mKed.
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WO 93/~24~3 2 1 3 5 0 7 2 Pcr/US93/0433~
~, Cells were suspended in Carnoy's, followed by several changes
c)f Carnoy's.
5 7. Thc cell suspension was dropped from a hai~ht of about 2 ft
onto humid slides and allowed to dry slowly in a humid
atmosphere.
8. Slides were a~ed overnlght on a 60C warmer before use.
a
i xample 2: Prepar~tion of Extended DNA Molecules
1. S~ells were to ~rown to canfluence on glass coverslips in
small petri dishes.
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2. The cuiture media was removed by aspira~ion and 0.075M KCI
with 0.01% Triton was ~dded, and cuWure Incubated for 20 rn at
37C.
2 0 3. KCI/Triton was removed, then cold chromosome isolation buffer
~1 .OM hexylene ~iycol, 0.5mi~A CaCI2, and 0.1 mM PIPES; Wray and
Stubi31efield ~e- Cell Res. 59:469-478 (1970)) wos ~ently
added and incubaied for 20m at 4C.
25 d" rhe buffer was removed, then a cold histone rernoving solution (0.2 mg/ml
aiextran sulphate, 0.02 m~/ml heparin, 10mM EDTA, 10mM Tris-HCI pH 9.0, 0.1%
Nonldet P-40, and l.OmM PMSF; Paulson and Laemn~li, Cell 12:817 828 (1977);
was added, and incuba~ed 30m at 4C.
30 5. ~ ~!ost of the so!ution (leav~n~ the ooverslip subrner~ed), was removed then 50%
Carnoy's flx (in dH20) was ~ently added, and allowed to stand for 5 m.
6. This solutlon ~as remcwed and the coverslip was aliowed to dry.
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7. The coverslip was flooded with Carnoy's, allowed to stand for 10 m and
followed with several chan~es o~ Carnoy's
B. The coverslip was removed and allowed to air dry.
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Example 3: 5~ Cosmnids from ~Q~ which Map to Human Chromosomes 18
and 21 to Demonstrate the Resolution A_hieved by the Methods
Described In ~1 and 2.
Maierial was propared as described in Examples 1 and 2.
Probss consisted of cosmids from a chromosome 18 or chromosome 21
conti~. :
Chromosome 18 co_ig: This conti~ maps to chromosome 18q23 and
comprises fi\/e cosmlcis spannin~ 10~ kb. This DNA molecule can be obtained fromthe American Type Cuture Collection (Rockville, i~lD) uncier Accession Number ~8~34
The 1wo flankins~ cosmlds (pWE7 (labelled with a ~reen fiuorophore)) and pWi-50w1
~labelled with a ~reen fluorophore) and the central cosmid (pWE50 (labelled with a
20 rad fluorophore)) were used In these experiments. Th~re is known to be a l~r~e ~ap
boh~/een pWE50 and pWE50w1; restriction di~ests indlcate th~t pWE7 and pWE50 andmay overiap.
. ~me 21 ~: This conti~ maps to chromosome 21 q22.3 anci
5 consists of five ~osmids extendin0 1 1 8kb. Thc two flankin~ cosmids (cHC 1-8A and pWE
18.3wl2) anai the Gentral cosmid ~pWE 19 2) were used In these experiments. Thisconti~ has been restrict~n mapped, and there is a 2.3 kb overlap of cHC1-8A with~: pWEI9.2 ~nd a 1.5 i-~b ~ap belween pWEI9.2 and pWE18.3w12.
30 i i All probes~;were labeled by nick translation. Hybridization cocktails
conta~n~d ~8 nç~/uL ot each flankin3 cosmld IcJbeled with digoxi~enin-dUTP of biotin
and ~8 n~juL of the central cosrnid l~beled with the altern~te hapten, 200 ng/uLhuman Cot-1 DNA, and 800 n~/uL of salmon DNA in 6XSSC, 10% Dextran Sulfate.
Supprossion hybr3dization and washin~ were essentially as described in Klin0er et. al.
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i: ." WO 93~22463 2 1 3 5 ~ ~ 2 PCl`/US93/04330
Am J. Hum Genet. 51(1): in press (199O, with the following rnodifications. The pre-
detection washes for ooverslips containin~ extended DNA molecules were: 1X~m
each In 2XSSC, 0.2XSSC, 0.1 XSSC; 3X~m in ~% formamide in 0.1 XSSC, 1 X5 in 2XSSC;
blocked with 3% i3SA/4XSSC for 5m. All aoverslips were washed at room temperature.
5 Hybridkation was detected with 0.5 u~/ml FlTC-antl digoxi0enin and 2.0 u~/ml Cy3-
streptavidin. Photo~raphs were taken directly frorn the rnicroscope with Kodak Gol~
400 film usln~ FlTC--Texas Reid (Qmega Optlcal~ or FITC-TRITC (Chroma Technology)
dual band pass fllters. Usln~ this labellln~ and detection scheme, the expected
pattern of fluorescence would be red-~reen-red or ~reen-red-~reen.
Re~uiHs
Rssc)lution of about 70kb was consistently ~chieved with butyrate ~reated
rnaterial and adJacent cosmids (3~40kb) were occasionally resolved. By ~relaxing~
15 Interphase chromatin, sodium butyrate rendered it more accessibie to hybridization.
These prep~ratlons were very claan; c~opla~m was rarely associated with the nuclei
or metaphase spreads~ Metaphase chromosomes typically appeared ~fuzy~ in these
preparations, su~gestln~ the presence of loops of decondensed chromatin which
made them less appealin~ for chromosome mappln~, but indlcated that butyrate
20 had the expected effect of decondensin~ the ohromcltin.
Hybridizatlon was consistently detected on extended DNA moleoules~ In
the more extended filaments, the si~nal appeared as a strin~ of fluorescent spots,
althou~h a considerable variation in the de~ree of condensation among the
hybridked molecules as well as withln the molecule was noticed. In one trial, the
25 antire len0th of the ~reen-reâ-~reen pattern representing the 18 contig was about
40um: the expected len~th for 109kb of B-DNA is about 37um. The three cosmids
could be unlquely Identified in these prep~rations. Consistent pa~terns of
fluorescence (representin~ the hybridized cosmids) were identified, especiaily where
the cosrnids met. Overlappin~ or abutting cosmids tended to run to~ether, wherea30 non-fluoresence ~aps were conspicuous.
213S072 f --
WO ~3~22~63 PCI /US93/04330 ` :
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A iar~e ~ap was evident between one red-~reen pair, consistent wHh the
relatior)shlp between pWE50 and pWE~wl.~ ~he other red-~reen pair appeared to becontinuous. This ~enerai patternj~of~jlaJ'~e ~ap between one red-~reen pair and a
sm~ll or no ~ap between the othe`r was consistently observed. This resolution enabled
s the determinc:~tion of absolute order of the three cosmids.
The three cosmids from the chrornosome 21 conti~ were accurately
resolved by hybridizatlon to extended DNA molecules. The 2.3kb overlap and the
1.~kb ~ap could be ~dentified in many of the filaments.
1 0 Equih~alents
Those skllled In the art will recognize or be able to ascertain using no more
than routine experimentation many equivalents ta the specHic embodiments of the
-
invention described specifically herein. Such equivalents are intended to be
15 encompassed In the scope of the followin~ claims.
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