CA2135072A1 - Methods for high resolution gene mapping - Google Patents

Abstract

Two convenient methods for ordering discrete DNA sequences at a high resolution are described. In one embodiment, the invention features a) contacting a eukaryotic cell containng a chromosome of interest with an agent that effects decondensation; b) preparing the cell containing decondensed chromatin for hybridization; and c) hybridizing the cell produced in step b) with at least two DNA
probes, each probe being complementary to at least one discrete DNA sequence present on the chromosme of interest; and d) detecting the presence and relative order of the probes as an indication of the relative and absolute locations of the DNA sequences on the chromosome. In another embodiment, the invention features ordering at least two discret DNA sequences on a fragment of DNA that has been extended, for example by physical means, chemical means or a combination of both. A preffered physical means for extending DNA is by gentle smearing.

Description

~ 2135072 ;.. 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 :: -: ~ ` :

2l3sn72 ;~ '"
093/22q63 ' : ~ PCr~US93/04330 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 ^ 2 -~ !
WO 93/~463 2 1 3 S 0 7 2 PCr/US93/04331) 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 . . .

. ~: : : , , ~ :

.

~ ~ .

WO 93/2~4S3~ 1 3 5 0 7 2 ; `; Pcr/US93/0433o `

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%
. .

;:

: ~ :

~ ~ ' ; 21~iB7~ !
:~ .` ..! WO 93/22463 PCI /US93/04330 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).

~ .
'` ~ ' :' .

3 2 1 3 5 0 7 ~ P~'r/US93/04330 ~ . i 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 : :
1. Short term Iymphocyte cuitures were establisheci usinçj standard ~ 2 0 protocols and incujated for 72 hrs c~t 37C.
: `
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.
:~ . .

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).
: ~

4. Cells were resuspend0d in BT/PMSF.(BT buffer plus 0.5mM
i30 ~ phenylmethytsu~fony!fluoride(PMSF)).
~'~
~ 5. Celis were resus;~ended in i3T buffer, then several drops af : ~
Carnoy's fix (3 parts methanol: 1 part acetic acld) was acided ;; ~ ~ to the celi suspenslon and gently mKed.

~ :

.
`` ~

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.
;i 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.

.
: ~

. -VV0 93/224n3 2 1 3 5 0 7 2 ~ Pcr/ us93/o4330 ( 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.
~ t 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.

.

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 ` :
.

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.

.
: : :
:' .

` ~: : .'

Claims (17)

1. A method for ordering at least two discrete DNA sequences on an interphase chromosome of interest comprising the steps of:
a) contacting a eukaryotic cell containing an interphase chromosome of interest with an agent that effects decondensation, thereby producing decondensed chromatin:
b) preparing the cell containing relaxed chromatin for hybridization:
c) hybridizing the cell produced in step b) with at least two distinct labelled DNA process, each probe being complementary to at least one discrete DNA sequence present on the chromosome of interest:
and d) detecting the presence and relative order of the probes as an indication of the relative and absolute locations of the DNA
sequences on the chromosome.

2. A method according to Claim 1 wherein the agent that effects decondensation is a salt of a short chain fatty acid.

3. A method according to Claim 1 wherein the agent that effects decondensation is a salt of a short chain fatty acid.

4. A method for ordering at least two discrete DNA sequences on a fragment of DNA, comprising the steps of:
a) smearing a fragment of DNA; thereby produicng smeared DNA;
b) preparing the smeared DNA for hybridization:
c) hybridizing the DNA produced in step b) with at least two distinct labelled DNA probes, each probe being complementary to at least one discrete DNA sequence present on time chromosome of interest;
and d) detecing the presenece and relaive order of the probes as an indication of the relative and absolute locations of the DNA
sequences on the fragment of DNA.

5. A method of Claim 4, wherein step a), the smearing of the DNA is accomplished by physical means.

6. A method of Claim 4, wherein step a), the smearing of the DNA is accomplished by chemical means.

7. A method for ordering at least two discrete DNA sequences on an interphase chromosome of interest, comprising the steps of:

a) contacting a eukaryotic cell containing an interphase chromosome of interest with an agent that causes histone hyperacetylation, thereby producing decondensed chromatin:
b) preparing the cell containing the decondensed chromatin for hybridization;

c) hybridizing the cell produced in step b) with at least two distinct labelled DNA probes, each probe being complementary to at least one discrete DNA sequence present on the chromosome of interest;
and d) detecting the presence and relative order of the probes as an indication of the relative and absolute locations of the DNA
sequences on the chromosome.

8. A method of Claim 7, wherein the agent that causes histone hyperacetylation is a salt of a short chain fatty acid.

9. A method of Claim 8, wherein the salt of a short chain fatty acid is selected from the group consisting of sodium butyrate, sodium propionate and sodium valerate.

10. A method of Claim 8, wherein the salt of the short chain fatty acid is sodium butyrate.

11. A method for locating at least two discrete DNA sequences on an interphase human chromosome of interest comprising the steps of:

a) contacting a human cell containing an interphase chromosome of interest with an agent, that causes histone hyperacetylation, thereby producing decondensed chromotin:

b) preparing the cell containing the decondensed chromatin for hybridization:

c) hybridizing the cell produced in step by with at least two distinct labelled DNA probes, each probe being complementary to at least one discrete DNA sequence present on the chromosome of interest:
and d) detecting the presence and relative order of the probes as an indication of the relative and absolute locations of the DNA
sequences on the chromosome.

12. A method of Claim 11, wherein the agent that causes histone hyperacetylation is a salt of a short chain fatty acid.

13. A method of Claim 12, wherein the salt of a short chain fatty acid is selected from the group consisting of sodium butyrate, sodium propionate and sodium valerate.

14. A method for ordering at least two discrete DNA sequences on an interphase chromosome comprising contacting a eukaryotic cell containing an interphase chromosome with a histone removing agent and analyzing the cell by fluorescence in situ hybridization using at least two distinct labelled probes which are complementary to at least two discrete DNA sequences on the chromosomes.

15. A method for ordering at least two discrete DNA sequences on a fragment of DNA, thereby producing smeared DNA and analyzing the smeared DNA by fluorescence in situ hybridization using at least two distinct labelled probes which are complementary to at least two discrete DNA sequences on the smeared DNA.

16. A method of Claim 2, wherein the salt of a short chain fatty acid is sodium butyrate.

17. A method of Claim 8, wherein the salt of a short chain fatty acid is sodium butyrate.