CA2170951A1

CA2170951A1 - Method of determining the presence and quantifying the number of di- and trinucleotide repeats and an instrument and kits thereof

Info

Publication number: CA2170951A1
Application number: CA002170951A
Authority: CA
Inventors: Nir Navot; Nurit Eyal
Original assignee: DIAGENETICS GAMIDOR LTD.
Current assignee: DIAGENETICS GAMIDOR Ltd
Priority date: 1996-03-04
Filing date: 1996-03-04
Publication date: 1997-09-05

Abstract

A method aimed at the quantification of di- and trinucleotide repeat which includes (a) treating a sample containing the nucleic acids of interest toobtain unpaired nucleotide bases spanning the position of the repeats and flanking regions, if the nucleic acids are not already single stranded; (b) contacting the unpaired nucleotide bases with an oligonucleotide primer capable of hybridizing with a stretch of nucleotide bases present in the nucleic acid ofinterest preferably 3' of the trinucleotide repeats to be quantified, so as to form a duplex between the primer and the nucleic acid of interest; (c) providing means to ensure that the examined nucleic acid and the oligonucleotide primer are confined to a reaction chamber at all further steps; (d) contacting the duplex with a primer extension unit which is capable of base pairing with the first nucleotide base in the core sequence of the repeats, and a template dependent extension enzyme; (e) eliminating non-incorporated primer extension units; (f) contacting the template primer duplex with a primer extension unit which is capable of base pairing with the second nucleotide base in the core sequence of the repeats, and a template dependent extension enzyme; (g) eliminating non-incorporated primer extension units; (h) contacting the template primer hybrid with a primer extension unit which is capable of base pairing with the third nucleotide base in the core sequence of the repeats; a detection moiety containing, primer extension unit which is capable of base pairing with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the trinucleotide repeats; and a template dependent extension enzyme; (i) eliminating non-incorporated primer extension units; (j) detecting for the presence of detection moiety containing primer extension unit; (k) steps(d) to (j) are repeated until detecting said detection moiety; (l) the number ofrepeats as stated under (k) enables the determination of the number of trinucleotide repeats, therefore enabling determination of the exact repetition number.

Description

~_ 2170~1 APPLICATION FOR PATENT

~v~lltols: Nir Navot and Nurit Eyal Title: A MEl~IOD O~ DETERMIN~NG THE PRESENCE AND
QUAN~IFY~G THE NU~ER OF DI- AND
TRINUCLEOTIDE REPEATS AND AN INSTRUMENT AND
KITS THEREFOR

lO This is a con~iml~tion in part of U.S. patent Application No. 08/084,505 filed July 1st, 1993, which is a con~inll~1;on in part of U.S. patent Application No.
07/919,872 filed July 27th, 1992, now abandoned.

~l~LD AND BACKGROUND OF THE INVENT~ON
The present inYenhon relates to ~e detell,lination of di- and ~inucleotide repeat mutations in~rolved with increasing number of geneticslly inherited diseases characterized by the expansion or the amplification of a core di- or trinucleotide sequences.
More particularly, the present inven~on concems a sensitive method, an automated ins~ument and kits for unequivocally quantifying the exact number of di- and trinucleotide repeats in preselected geneac loci.

2s The present method, ins~urnent and kits are also useful for ~ete~.. ;.~; ~g the number of head to tail repeat sequences of two or more nucleotide bases, provided that the core sequence consists of no more than ~ree types of nucleotide bases such æ, for example, ~enine (A), guanine (G) and cytosine (C) in a core sequence composed of (AAGCGCA)n (n 2 1).
In recent years, increasing number of genetically inherited diseases were-found to be associated with mutations desigrated unstable trinucleotide repeat mutations in which a core sequence of three nucleotide bases is exp~n~e~ or amplified, such that affected individuals and in some cases carriers, co.l~ai 3~ more repe~ an a~)p~elld~r healthy ones, in dle particular DNA locus implicated with the disease.

2170~Sl .~ 2 More r~ce.~ , a new cancer gene was discovered and wa~s shown to cause segJnentC of DNA to be abnorm~11y l~pe~l4~ in pairs and/or triplets in tumor cells of individuals caT~ying the cancer gene. In this case hundred of thousands of short units of DNA are copied over and over again, pres...n~bly s destroying the tumor cells ability to control their growth. This phenomenon issomewhat di~elent than the e~ ion of u~Lstable tnnucleotide ~e~eals in L~c~ ce~ses since it occurs in many DNA loci within the same cell.
Nevertheless, there is a good reason to believe that both pher~om~n~ are p op~e~ by similar mech~nicmc. See, Kolata G. Health/Science section, The lO National Herald Tribune, Thursday, May 13, 1993.

So far, seven genetically tr~ncmitte~l ~lice~ces each involving a unique genetic locus, have been implicated with trinucleotide repeat mutations. These include: Fragile XA (A site, Mar~n Bell) syndrome (FRAXA); spinal and bulbar m11sc 11~r atrophy, SBMA (Kermedy ~ice~ce); Myotonic dystrophy (CursGhm~nn Steiner~ DM); H11ntington's disease (HD); Spinocerebellar ataxia type 1 (SCAl); Fragile XE (E site) m~nt~l retardation (FRAXE MR); and De.~to.ubral pallidoluysian atrophy (DRPLA). Since di- and trinucleotide l~peals have been observed within or close to a number of additional human 20 genes by gene-bank searches, it is conceivable that di- and trinucleotide amplifications may be involved in the c~1-s~tion of other genetic ~ice~ces as well.

Fragile XA syndrome is an X chromosome linked recessive disorder with 2s incomplete penetrance. It is characterized by moderate to severe mental r. t~ation and other phenotype characteristics, and is one of the most common forms of mental retardation with an estim~tç~l incitlPnce of 1 in 1250 males andcoll~s~onding 1 in 2500 females (heterozygotes), rendering this disease one of the most common human diseases and the most common form of f~mili~l 30 ~ tion. Fragile X chromosomes present their unique phenotype when leukocyte cells carrying them are grown in culture under folate starvation. As mentioned, ~he fragile X syndrome is char~cteri7~d by incomplete penetrance hence (l) some males, r~ f~l~d to as no~nal tr~smittin~ males (NTMs), are clinically nonnal but are infe.led to carry the genetic defect by a position in 3s pedigrees rendering them obligatory carriers; (2) one third of female caITiers have evidence of mild mPntql ;.Y~ Genetic 1ink~ge studies effected by restriction L~..~n~ Iength polymorphism analysis of illfolmative pedigrees; and 5omq~ic cell hybrid studies of hqmetçr chromosomes c~ying translocated ~ 3 se~entc of human fragile X chromosomes in cells grown in culture under folate starvation, enabled the loc~li7~tion of the fragile X gene to chromosomalband q27.3 on the X chromosome (Xq27.3). Eventually the fragile X defective gene, ~3esi~tÇ~ fragile X mental reta~dalion 1 (~MRl), was isolated via 5 positional cloning and led to the discovery of a highly polyrnorphic (CGG)n sequence within its 5' untr~nsl~te~ region. Population and fragile X patients scfeenillg revealed that healthy individuals are characterized by low numbers ofthe (CGG)n trinucleotide repeat (n = 6-52); carriers are characterized by metlillm nwnbers of the (CGG)n tnnucleotide repeat (n = 50-200); and affected o individuals are charactçri7e~ by high numbers of the (CGG)n l~inucleotide repeat (n = 230-1000). When the (CGG)n tnnucleotide repeats of the FMRl gene ex~-eeds app~o~;...~tely 230 repeats, the DNA of the entire 5' region of thé
gene l)eCOlUes abnorrnally methylated. This methylation ext~nds u~slle~n into and beyond the promoter region and results in the transcriptional suppfession oflS the FMRl gene leading to the cessation of the FMRl protein production which is probably the cause of the phenotype. See Annernieke J.M.H. (1991) Cell, 65:905-914; Pieretti M. (1991) Cell, 66:817-822; Caskey T.C. et al. (1992) Science, 256:784-788.

Spinal and bulbar muscular atrophy (SBMA), like the fragile X
s~ndrome, is a rare X linked recessive genetic disorder characterized by ~-3ulthood onset of progressive mllsc~ r weakness of upper and lower e ~ niLies which is secondary to neural degeneration. Affected males have re~lce~ fertility and excessive development of the male m~mm~ry glands 2S (gynecomastia); female carriers have few or no s~",-ptoms. Genetic linkage analysis of informative pedigrees enabled the loc~li7~tion of SBMA to chromosome Xqll-12, the region where the gene encoding the androgen feCeptO~ (AR) was previously loc~li7e~, rendering this gene a candidate for SBMA. Studies of the AR gene revealed a highly polymorphic (CAG)n trimlcleotide repeat, sitll~te~i in exon 1, encoding a variable polyglu~ e stretch in the AR plole~ urther studies of the AR gene from normal and SBMA ~ect~d individuals revealed that while low numbers of the (CAG)n trinl~cleotide repeat (n = 12-34) characterize ap~alelllly healthy individuals, high numbers of the (CAG)n trinucleotide repeat (n = 40-62) char~cteri7e 3s SBMA ~,cled individuals, while a carrier state is not yet known for this mnt~tion The infll)pr~ee of the e.~.An-lecl pol~gl,~ .ine tract on ~he AR prote~is not yet established, nevertheless, gain of function, le~tlin~. to ~e SMBA

. 2170951 ~_ 4 phenotype is ~uspecled. See, Albert R et al. (1991) Nature, 352:77-79; Caskey T.C. et al. (1992) Science, 256:784-788.

Myotonic dy~ opl~r (DM) is an autosomal do~ina~t ~ice~ce s characterized by myotonia, cardiac arrhythmi~c, cataracts, male balding, male infertility (hypogon~licm)~ and other associated endocrinopathies. The rare con~nit~l form of DM is associated with profound newborn hypotonia and m~nt~l ret~rd~t;on. DM has a prevalence of 2.5-5.5 affected per 100,000 individuals. DM was mapped by genetic linkage to chromosome l9ql3.3 and o the DM gene, ~çsign~te~ myotonin protein kinase (MT-PK), was isolated via positional cloning and other molecular metho.ls. Further studies revealed a polyrnorphic (GCT)n trinucleotide repeat situated in the 3' untr~ncl~te~ region of the MT-PK gene. Analyses of the MT-PK gene from normal and DM
~ccled individuals revealed that while low numbers of the (GCT)n lS trinucleotide repeat (n = 5-37) characterize ap~ elltly healthy individuals, high mnnb~r5 of the (GCT)n trinucleotide repeat (n = 100 - >1000) characterize DM
affected individuals, while the carrier state is characterized by medium numbersof the (GCT)n tnnucleotide repeat (n - 50-100). Further studies have revealed that expansion of the (GCT)n t~inucleotide repeat leads to increased MT-20 PKmRNA stability, therefore to the production of more MT-PK protein susl.cctedly le~ling directly or indileclly, to the DM phenotype. See, Fu Y.H.
et al. (1992) Science, 255:1256-1258; Caskey T.C. et al. (1992) Science, 256:784-788.

2s H~ tin~on's ~ice~se (HD) is a devastating late onset autosomal domin~rlt neurodegenerative disorder characterized by progressive neurodegeneration with personality dis~ ce, involuntary movements, cognitive loss and an inexorable progression to death 15-20 years from time of onset. HD occurs with a frequency of 1 in 10,000 individuals in most populations of C~lc~si~n ~escr-~-t The HD gene was loc~li7ed to chromosome 4pl6.3 by genetic linkage analysis with polymorphic DNA markers. Recently, following 10 years of s;~e rcsed.ch, the defective gene c~lsing HD, ~es~ te~ IT15, was isolated and a polymorphic (CAG)~, trinucleotide repeat encoding a poly~l~J~ e stretch, s;~v~te~l in exon 1 of the gene was discovered. It was 3s further found that the (CAG)n trinucleo~de repeat is P~p~n~e~ in HD
chromoso-..es (n = 42-100) as co...l.~. ed with no~nal chromosomes (n = 11-36), pl. s ~ bly leading to IT15 ~-ot u~s gain of function, suspcdcdly lç~ling to theHD ~hen~,~ype. See, The H~mtin~on's di~e~se collabora~ve lcse~ch group 2170~51 . . , . ~_ 5 (1993) CelL 72:971-983; 7luhlk~ C. et al. (1993) Hum. Molec. Genet. 2:1467-1469.

Spinocerebellar ataxia type 1 (SCAl) is a progressive late onset 5 autosomal ~omin~nt disorder characterized by ataxia, ophthalmoparesis and vanable degree of motor we~knçss due to neurodegeneration of the cerebellum, spinal chord and brain stem, leading to complete disability and death 10-20 years after onset. The SCA1 gene was loc~li7e~ to chromosome 6p22-p23 due to strong genetic !inkage with the highly polymorphic HLA locus and other 0 polymorphic DNA markers. The defective gene c~usin~ SCAl, was isolated in a yeast artificial chromosome contig and subcloned into cosmids. A polymorphic (CAG)n trinucleotide repeat encoding a polygl~ -nine stretch, situ~te~ in exon 1 of the SCAl gene was discovered. It was further found that the (CAG)n trinucleotide repeat is exp~ndefl in SCAl chromosomes (n = 43-81) as co~ ared wi~ normal chromosomes (n = 19-36), presu,nably leading to SCAl prot.,~s gain of function, suspectedly leading to the SCAl phenotype. See, Orr H.T. et al. (1993) Nature Genetics, 4:221-226.

Fragile XE mental let~rdalion (FRAXE MR), like FRAXA is an X
20 chromosome linked recessive disorder with incomplete penetrance. It is c~acte.i;ced by moderate to severe mental retardation and other phenotype characteristics. Like FRAXA, FR~XE chromosomes present their unique phenotype when leukocyte cells carrying them are grown in culture under folate starvation. Genetic linkage studies enabled the loc~li7~tion of the FRAXE gene 2s to chromosome Xq28. Eventually the FRAXE gene was isolated via positional cloning and led to the discovery of a highly polymorphic (GCC)n trinucleotide repeat segrep, ~ with the llise~se. Population and FRAXE patients screening revealed that healthy individuals are characterized by low numbers of the (GCC)n trinucleotide repeat (n = 6-25); carriers are characterized by medium 30 numbers of the (GCC)n trinucleotide repeat (n - 116-133); and affected individuals are characterized by high numbers of the (CGG)n trinucleotide repeat (n = 200-850). When the (CGG)n trinucleotide repeat of the FRAXE
gene exceeds appro~ tely 200 repeats, the DNA of a CpG island located in the trinucl~tide lepedls vicinity becomes abnormally methylated, presu",ably 35 leading to the secescion of the FRAXE ~rote,l, production, which is probably ~he cause ofthe plle~ol~rpe. See Knight S.J.L. et al. (1993) Cell, 74:127-134.

Delltalolubral pallidoluysian atrophy (DRPLA) is a late onset autosomal do...;~ .t ntulodege~erative disorder, prevalent in Japan, characterized by a varying combinations of progressive myoclonus, epilepsy, ataxia, choreo~thetosic and d~menti~ Neuropathological changes consist of combined ~l.eg~nf~ration of the ~e-.tAtc,l~lbal and pallidoluysian systems of the centralnervous system. The ~1ise~se is further characterized by variable penertance, even in a single family. T.ink~e analysis in DRPLA families enabled to localize the DRPLA gene to chromosome 12pl2-13. The DRPLA gene was isolated via - scree.-.ng for (CAG)n unstable trinucleotite repeat that was found to be located o in exon 1 of the gene, encoding a variable polygl~ ;ne stretch in the DRPLA
protein. It was further found that the (CAG)n trinucleotide repeat is expanded in DRPLA chromosomes (n = 49-75) as comparcd with normal chromosomes (n =
7-23), presllm~bly le~ding to DRPLA ~lole~,s gain of function, suspectedly leading to the DRPLA phenotype. See, Nagafuchi S. et al. (1994) Nature lS Genetics, 6:14-18; Koide R. el al. (1994) Nature Genetics, 6:9-13.

Because of the high frequency, variable penetrance and instability of Fragile XA syndrome and other genetically inherited disorders associated with ~imlcleotide repeats e~p~ncion~ there is a widely recognized need for, and it 20 would be highly advantageous to have, a low cost method, demanding merely non skilled personnel for its execution, that enables the efficient and accurate~lete....i~-~tion of the number of repeats in various genes.

Unlike the cornmon gene mutations (e.g., Cystic Fibrosis /~F508), which 2s are stable, that is, they are transmitted nnch~n~ed along the generations of pedigrees, the situation is somewhat di~erent for the trinucleotide repeat mutations which are charactcl;,e~l by instability, that is, when the number of repeats exceeds a threshold value, these mutations have a tendency to expand and inclllde a greater number of repeats (1) when vertically tr~nsmitted from 30 yale~ to children along genetic traits; and (2) when somatically transmitted to ght~r cells iTl a given individual, a phenomena ~esiQn~ted somahc instability, yielding mosaicism.

The two ~pes of instability chalactelizing tTinucleotide repeat mutations 3s will be exemplified herein for the fragile XA syndrome.

Fragile XA unstable alleles are observed in norm~ A~ g males (NTMs) their as~ to.l,atic ~ ghters and s~ o,nalic male gran~l~hilds.

2170~51 When the number of trinucleotide repeats of such alleles was determined, it was found to increase along generations, in one example from 82 in the NTM father to 83 in the as~tQ~ tic d~v~hte~ (90 in a second as~ )to~--At;c ~n~ht~r) to >200 in the ~ise~seA gr~ndcllildren. The 82, 83 and 90 repeats cor.~ i..g alleles s are refe.l~d to as pre~ ;on alleles. It was a study of numerous families of this type that pc...~;Ued a correlation of the phenomenon of anticipation (earlier ages of onset or severeness in sl)ccessive ge~elalions) and the molecular events of the (CGG)n eYp~ncion. NTMs carry numbers of CGG repeats outside the range of normal and bellow those found in affected males. Such males transmit the 0 rc~eals to their prog.,~ with relatively small changes in the repeats number. on the other hand fem~les who carry similar ~re.r...l~;on alleles are prone to bearplOge~t (male or female) with large e~cr~nsion of the repeats region. Thus, large CGG amplification associated with fragile XA syndrome appears to be pre~lon~inA~ a female meiotic event. See, Caskey T.C. et al. (1992) Science, 256:784-788.

Many fragile XA ~liceace~3 individuals were found to be mosaic in respect with 1he number of the CGG trinucleotide repeats characterizing different cells in their body, a phenomenon in~lic~ting somatic instability of expanded repeats.
~ stability, characterized by expansion of trinucleotide repeats, is observed also in DM, HD, FRAXE, DRPLA and SCAl pedigrees. As opposed to E~E~AXA. DM and FRAXE high risk alleles can expand to similar extent via both male and female meiosis and to the best of our knowledge somatic 2~ mosaicism wa not yet observed in DM and FRAXE patients. High risk alleles were not yet found for HD and DRPLA, that is, alleles of these lise~ses are either ca~ying or not canying the flice~se Never~eless, HD repeats are also unstable in more than 80% of meiotic tr~ncmi~sions but, on dle other hand, they are c~a~aclelized by increasing, or alternatively, decrea g numbers of repeats 30 wi~ ~he largest illcr~ase occullulg in paternal tr~ncmicsion (Duyao M. et al.(1993) Nature Genetics, 4:387-392), whereas DRPLA alleles have a tendency to increase in size along generations. See, Nagafuchi S. et al. (1994) Nature Genetics, 6:14-18; Koide R. et al. (1994) Nature Gene~cs, 6:9-13.

3s All~,l~ts to correlate the size of trinucleotide repeat mutations and the sevent~r of t~e ~csoc;s~ netic 3ice~ces were made for f~agile XA syndrome, myotonic d~a~o~h~ e~.~A~ulubral pallidoluysian allo~Ly and spinocerebellar ataxia type 1.

2170~1 For fragile XA, as expected, median IQ score was significantly lower for fem~les carrying a fully eYp~n~e.l mutation (above 230 repeats) than for females carrying a plf~ ;on (50-200 repeals) on one of their X
s chromosomes On the other hand, no significant relationship was found between IQ score and number of CGG repeats, see, Taylor A.K. et al. (1994) JAMA, 271:507-514. Never~heless, it was found that prenatal DNA studies of the number of CTG tnnucleotide repeats characte~ g myotonic dystrophy alleles can improve the estim~tion of clinical severity; and that the number of CAG
0 trinucleotide repeats in spinocerebellar ataxia type 1 and de~ tol ubral pallidoluysian atrophy is correlated with increased progression of the disease (N~g~filc-lli S. et al. (1994) Nature Genetics, 6:14-18; Koide R et al. (1994) Nature Genetics, 6:9-13; Orr H.T. et al. (1993) Nature Genetics, 4:221-226).

A~ ls to correlate between the size of trinucleotide repeat mutations and the age of onset of H~mtin ton's disease resulted in finding a reversed correlation confined to the upper range of trinucleotide repeat nurnbers (ca. 60-100 le~eats), see, Andrew S.E. et al. (1993) Nature Genetics, 4:398-403.
Fu~ P .nore, for spinocerebellar ataxia type 1 and ~e~ tolubrâl pallidoluysian 20 allopLy (Nagafuchi S. et al. (1994) Nature Genetics, 6:14-18; Koide R. et al.(1994) Nature Genetics, 6:9-13), â direct correlation between the number of the (CAG)n trinucleotide repeats expansion and earlier ages of onset was found.

Collectively, these data call for the development of a reliable, accurate 2s and easy to operate di- and trinucleotide repeats quantification method aimed at post and prenatal ~ ~osis and prognosis.

Three basic methods are Cl~lenlly used to ~ete~mine the number of di-and trinucleotide r~peat~ in any particular locus, these are: (1) "Southern" blot 30 analysis; (2) in vitro amplification via the Polymerase Chain Reaction (PCR) and PCR fra~rnPnt size ~letermin~tion; (3) DNA sequencing (usually of PCR
amplified fr~ e-.ls).

"Soulllell," blot analysis for the quantification of di- and tnnucleotide 35 ç~eals is a method based upon: (1) enzymatic cleavage of genomic DNA
ol,la;"ed from ~he e~..;..e~l individual via sequence specific restriction enymes cleaving the DNA at many sites including ~e fl~nking regions both 5' and 3' to the DNA region co..l~;n;..g the ~ ;..ed di- or trinucleotide repeats;

2170~1 ~ g (2) gel electrophoresis aimed at size separation of the DNA fragments obtained under step (l); (3) blotting or transferring the cleaved and size separated DNA
fr~nPntc to a test surface; (4) ~rep~ a labeled probe capable of specific hybridization with the blotted DNA fragment co..~ g the repeats; (5) 5 hybri~li7ing the labeled probe with the blotted DNA fragments; (6) washing offprobe excess to obtain specific hybri-1ization and to reduce non-specific and background sigr ~lc; (7) ~etecting positive signals via means dependent upon theprobe labeling ter-~mi~lue employed under step (4); (8) illte.~-cting the results by ~ete- -.inin~ the size of the fragment hybridized to the labeled probe; and finally o (9) calc~ tin~ the number of di- or tnnucleotide repeats.

"Soulhe.ll" blot analysis for the quantification of di- and trinucleotide repeats has major drawbacks: (1) the method is primarily dependent upon the eYict~-nce of suitable restriction enzymes recognition sites in the immediate 5'lS and 3' fl~nking region of the repeats region; (2) gel electrophoresis employed under "Southern" blot analysis has low resolution capacity for small size variations, therefore this method is not suitable for monitolil g small variations in the number of the di- or trinucleotide repeats; (3) "Southern" blot analysis is not capable of ~ictin~lishing between size variations due to di- or trinucleotide 20 repeats expansion/de-expansion or other molecular events such as the loss or the formation of a restriction enzyme recognition site/s due to point mutation, deletions or insertions, yielding a di- or trinucleotide repeats expansion/de-e~ansion independent length polymorphism; (4) "Southern" blot analysis dem~n~ls acculate exec-~tion of a multistep procedure, of which most steps 2~ include several complicated steps, are tirne consuming and require highly skilled personnel for their routine execution, especially gel electrophoresis, blotting and hybridization; (5) highly skilled personnel are also needed for intel~leling theresults and for calc~lh~ing the di- or trinucleotide repeats number; (6) gel elecllophoresis, hybridization and washing conditions may vary considerably 30 depending upon fr~nPnt size and sequence, therefore, "Southern" blot analysisr~q ~ircs dilre~,lt calibration of the procedure for any given ~lise~ce; and last but not least (7) due to its being a mnl1i$tep procedure, "Southern" blot analysis is not easily applicable for complete autom~ti7~hon.

In vi~ro amplification via the Polymerase Chain Reaction (PCR) and PCR fr~nen~ size detelll~ation is easier for ex~cnhon as colllp&~d with "soulhelll" blot analysis and involves less steps, these are: (1) PCR
~m~!ification of the di- or trinucleotide ~eal~ region using PCR plilllCIs ~om 217095i the 5' and 3' fl~nkin~ regions of the repeats; (2) size ~letçrrnin~tion of thus ot~t~ined PCR fragments via high resolution gel electrophoresis; and (3) c~lcnl~tin~ the number of the di- or trinucleotide repeats. See, Erster S.H.
(1992) Hum. Genet., 90:55-61.
s Although this approach is simpler and therefore easier for routine eYeclltion it shares some of the drawbacks described for "Southern" blot analysis, these incl~lde (1) the PCR approach is not capable of ~listin~lichin~
bet~veen size variations due to di- or trinucleotide repeats e~p~nsion/de-0 eYp~n~ion or other molecular events such as the loss or gain of sequences due todeletions or insertions in the 5' and/or 3' repeats fl~nkin~ regions, yielding a di-or tnnucleotide repeats expansion/de-expansion independent PCR fr~rnent length polymorphism; (2) a high resolution gel electrophoresis is required for resolving small size var,iations in the PCR fr~rnents, this calls for hi~hly skilled s personnel and therefore not suitable as a routine diagnostics procedure; (3) highly skilled personnel are also needed for illte.~le~ g the results and for c~lc~ tin~ the number of the di- or trinucleotide repeats. In addition: (4) the PCR approach is not suitable for quantifying highly expanded di- or tlinucleotide repeats, since its amplifying capacity is limited to relatively small 20 ~n.ontc, therefore, in cases where the fragment to be amplified exceeds a certain size limit the PCR reaction will fail to yield a specific product; and (5) some of the di- or trinucleotide repeats fonn highly GC rich stretches of DNA
which are not easily amplified via standard PCR protocols.

2~ The most basic method for ~e~ .. ;n~tion of di- or trinucleotide repeats number is DNA sequencing. The most widely used sequencing method is based on the dideoxynucleotide chain termin~tion procedure. The technique involves the incorporation of dideoxynucleotides with the aid of a DNA extension en~me at the 3'-end of an elong~inf~ DNA chain. Once the dideo~ynucleotide 30 has been incol~olated, f~er elongation of dle chain is blocked. See, Sanger F.
(1981), Science 214, 120~ 1210. Recently automated DNA sequencing techniques have been developed which provide for more rapid and safer DNA
seql-entcin~ One such approach utili7~s a set of four chain terrnin~tin~
fluor~scçntly labeled dideoxynucleotides. See Chehab, F.F., et al. (1989), Proc.3s Na~d. Acad. Sci. (USA) 86, 9178 9182; Prober, J.M., et al. (1987), Science 238, 336 341; Smith, L.M., et al. (1980), Nature 321, 674 678). In this metho~
succinyl fluo,.,sce~n dyes are used. Each dideoxynucleotide receives a dif~lent dye of di~,ellt al)sol~Lion and emission characteristics. Thus, DNA molecules l~bele-l with each of the di~cre,lt dideoxynucleotides may be ~ tin~ hed from one another. Using these dideoxynucleotides, it is possible to sequence a DNA
seg~f-.t by carrying out a single reaction in which all four of the di~rerellllyl~bel~d dideoxynucleotides are added together into a single reaction llu~ , and s the res~llting labeled oligonucleotide fr~n~nte may then be resolved by polyacrylamide gel electrophoresis in a single sequencing lane on the gel. The gel is then sc~nne~l by a fluorimeter capable of distingnishing the di~rerellt fluorescent labels. The sequence of the diLrerent labels along the lane is then tr~ncl~ted into ~e sequence of the tested DNA se~rnP-nt DNA seq~lencing as a method for ql-~ntification of di- or trinucleotide repeat numbers has few major drawbacks, these are: (1) a high voltage and high resolution gel electrophoresis is required for resolving the single stranded DNAnested fragments obtained during the sequencing reaction, differing from each 5 other merely by one nucleotide base, this calls for highly sk~led personnel and therefore not suitable as a routine diagnostics procedure; (2) some of the di- or ~mlcleotide repeats form highly GC rich stretches of DNA which are not easily sequenced via standard sequencing protocols; and (3) the sequencing approach is not suitable for quarl~ir~ying large di- or trinucleotide rcpedls since it is limited 20 by the resolution power of the sequencing gel.

It is an object of the present invention to provide a simple, reliable, rapid, highly accurate and easy to operate di- and trinucleotide repeats quantificationm~,~o~ aimed at post and prenatal diagnosis and prognosis which do not require 2s electrophoresis or similar separation according to size as part of its methodology.

It is another object of the present invention to provide a diagnostic kit and an automated instrument to be used for carrying out the above method of 30 the invention.

2170~51 ` 12 SUMMARY OF THF. ~VENTION

Acco,ding to the ~resenl invention, there is provided a method, named Trinucleotide Repeats Quallti~ication (TriQ), for ~ete-,.,;.-in~ the number of di-s and trinncleotide repeals associated with various liise~ses.

The method of the invention dep~o-n~ls upon counting s~ccessive steps of incorporation of two to three types of primer extension units, depending on the core repeat sequence, to a 3'-end of an oligonucleotide primer annealed to a 0 sin~le stranded nucleic acid sequence template preferably 3' of the di- or trim~clsotide repeats region, said counting being terrnin~te~ at the incorporation of an additional type of primer ext~oncion unit, cont~ining a detection moiety, capable of base pairing unth a nucleotide base located 5' of the repeats region,said nucleotide base is the first nucleotide base that is not identical with thes nucleotide bases in the core sequence of the repeats in said nucleic acid template.

One of the applications of the current invention is to enable quick ~lGte-,..;..~l;on of the number of di- or trinucleotide repeats in genetic loci 20 co.~f~ such repeats.

In the broad application of the method of the invention the addition of prirner eYt~ncion units to a 3'-end of the oligonucleotide plimer is carried outsin~ rly, that is, primer extension units complementary to the di- or 2s trinucleotide repeats core sequence are added at the 3'-end of the elong~ting oligonucleotide primer one after the other. Also according to the broad application of the method of the invention the detection moiety cont~ining p,ill1er eytçncion unit that is complelucnt~y to a nucleotide base located 5' ofthe repeat~ region, which is the first nucleotide base not identical with the 30 nucleotide bases in the core sequence of the repeats, is present in all-i,lcol~or~Lion steps.

In a plefe.le~ use of this application of the present invention for the ~et~ ;on of the mlmber of trimlcleotide repeats in genetic loci CO,,I~;"il~g 3s such lcp~t~, the addition of primer extension units to a 3'-end of the oli~om~cleQtide primer is carried out in pairs followed by a single primer P.l~ n unit addition, or ~lt~om~tively~ vice versa, a single primer extension unit ad~lition is followed by the addition of a pair.

-. 2170951 Also accor&lg to the plefelled use of this application of the method of the invention a detection moiety co..~ g primer eYtencion unit that is comple...e-.~ r to a nucleotide base located 5' of the repeats region, said nucleotide bace is the first nucleotide base not identical with the nucleotide bases in the core sequence of the repeats, is present only in some or all steps prcccA;n~ the incolyolalion of a primer extçncion unit complem~nt~ y to the first nucleotide base in the core sequence of the di- or trinucleotide r~eals.

0 Another use of this application of the cullenl invention enables genotyping of an individual, that is, to determine the genotype of an individualat any DNA locus co--~ g di- or trinucleotide repeats, that is to qua~ the number of re~eats cont~ined in any of said locus allele.

Yet another use of this application of the cull~,nt invention enables the dete....i..~tion of the number of head to tail repeat sequences comprised of twoor more core nucleotide bases, provided that the core repeat sequence consists of no more than three types of nucleotide bases.

According to features of prefe~led embodiments of the invention described below suitable detection moieties of primer extension units include those facilitating direct or indirect detection and which are pc~ anendy conjugated to any location at the primer extension unit, or alternatively, removable or destructible. Detecting dhe detection moiety, whether direcdy or 2~ indirectly, may be carried out in the reaction chamber or in a dirrelellt chamber depending whether the detection moiety is removable or not.

AccGrding to still further fealures in the described preferred embo-lim~nts, the e~erlcion moiety is a deoxyribonucleohde, such as dATP, dCTP, dGTP, dTTP and dUTP.

The oligonucleotide primer may be of any suitable length. Time and e~l ellsc considerations tend to shit yrefer~llce toward shorter oligonucleotide which is still sufficiently long to ensure high sequence specificity while at the 3~ same time e ~Iclll ;1l~ rapid, easy and accuratc l~reparalion.

The sample of genetic m~ten~l being tested by the method of invention may be in the form of RNA or DNA.

The e~tension moiety may, for example, be ~ rl-ed to any suitable dçtech-n moiety, such dS a radioactive label, e.g., 32p and various fluorescent labels. Another example involves nucleotides having a detection moiety 5 attacllment which may function for indirect detection.

Accord~g to furdler fealu~es of ~fe.l. d embo~lime~tc described below re~e,e..lc are collected and are reused in further steps.

Also accor~ing to the present invention, there is provided a diagnostic kit for quantifying the number of di- or trinucleotide repeats, concictin~ (a) any number of suitable oligonucleotide primers (b) two or three primer ç~tencion units; (c) further one or two primer extension units of a type not included under (b), said primer extencion UllitS co~ a detection moiety; (d) a template lS depen~lPnt extencion en~yrne; and (e) at least one buffer.

Also according to the present invention, there is provided an automated i,lsl~ ent suitable for executing the steps concistin~ the mel;hod of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with refclence to the accol,lpanying drawings, wherein:
2~
FIG. 1 is a scll~m~ic outline of fealules of a method aimed at q~ ir~ing the number of CAG trinucleotide repeats in examined nucleic acid sequence.

FIG. 2 is a schPm~tic oudine of features of modified and more efficient 30 methods aimed at ~ ir~ing the nllmber of CAG trinucleotide repeats in-,A.n;ne~ nucleic acid sequence.

FIG. 3 is a sçhPm~tic ou~dine of fealules of a method aimed at Cim~ eous q.~ ification of ~e number of CAG trinucleotide le~)edls in two 3s alleles each c~--4;--c a difrerent number of said l~peals.

FIG. 4 is a sr-h~ oudine of fca~ s of an i~l.u~nent suitable for eY~c~ the steps consistinp dle method of the inventio~

n~SCRIPTION OF THF. PREFER~F.n EMBODIMENTS

The present invention is of a novel method for dete~ninin~. t~.e number of 5 di- or trinucleotide lcpeals associated with vanous ~i~e~cec The principles and operation of a method according to the present invention may be better understood with leferc~ce to the drawings and the accG.~ying descliylion.

The plese.~t invention will be described in more detail with emphasis on a method for qu~ liLcation of the number of trinucleotide repeats in genes, which re~eals are associated with genetic disorders. While this application of the method of the invention is presently ylefe~led~ this is by no means the onlyapplication of the invention as will no doubt be appreciated by those skilled inthe art. For example, the method has various other applications including, but not li~ c.1 to, the detection of specific genetic sequences in samples such as nucleotide repeats characterized by a core sequence consisted of two or three types of nucleotides; those associated with certain genetic or other diseases and 20 pathogenic microorg~nisms for example bacteria and viruses; in testing of c-"i1~; and in forensic medicine; cancers; and plant polymorphism.

As for quantifying the nurnber of trinucleotide repeats in genes associated with trinucleotide repeats expansion the method of the invention 25 depends upon counting sllccessive steps of incorporation of two to three types of primer extension units, depending on the core repeat sequence, to a 3'-end ofan oligonucleotide primer annealed to a single stranded nucleic acid sequence template preferably 3' of the trinucleotide repeats region, said counting being telmin~te~l at the inco"~oralion of an additional type of primer extension unit,30 CO.~ p a det~ction moiety, capable of base pairing with the first nucleotide base of a type that is not included among the nucleotide bases in the core sequence of the repeat in said nucleic acid template.

In order to better unde~ d the embodiment of the present invention, 35 1~fc~ ce is made to Figure 1, which is a schematic depiction of a method aimed at quantiijring the numb~- of CAG trinucleotide ~pcals in e~A~..;.~e~ nucleic acid sequence which inclndes: (a) if ~e nucleic acid of inte~s1 is not single stranded, ~eating the sample cont~inin~ the nucleic acid to obtain u~lpaired - - 2170~51 nudeotide bases S~3A~ . the repeats and fl~nking regions; (b) under hybri~i7~tion conditions, contActing the ul~pai~ed nucleotide bases with an oligom~rleoti~e primer, having a sequence which is complçmçnt~ry to a stretch of nucleotide bases sit~l~te~ p~fe~ably 3' of the repeats region in the e~ ed s single strand sequence, ~I~;fe,ably, the 3'-end of said oligonucleotide primer is annealed to the first nucleotide 3' of the l~eals region; (c) providing means toensure that at least the ç~r~mined nucleic acid and the oligonucleotide primer are . confined to a reaction ch~mber at all further steps; (d) the template primer hybrid is contacted with a primer extension unit which is capable of base o pairing with the first nucleotide base in the core sequence of the repeats, dGTP
in the given example, and a template dependent extension enzyme; (e) after a sl~itable incllbation time, non-incorporated primer extension units are climin~teA preferably washed away; (f) the template primer hybrid, now said primer elongated by one unit, is contacted with a primer extension unit which islS capable of base pairing with the second nucleotide base in the core sequence of the repeats, dTTP in the given example, and a template dependent extension enzyme; (g) after a suitable incubation time non-incorporated primer extension units are elimin~te~l p~efelably washed away; (h) the template primer hybrid, now said primer elongated by one additional unit, is contacted with a primer 20 e~rt~n.cion unit which is capable of base pairing with the third nucleotide base in the core sequence of the re~eal~, dCTP in the given example; a detection moiety co.~t~i..in~ primer extension unit which is capable of base pairing with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core 2~ sequence of the trinucleotide repeats, dATP$ in the given example; and a template dependent extension enzyme; (i) after a suitable incubation time non-incorporated primer extension units are elimin~te~ preferably washed away; (.i) detccling for the presence of the detection moiety cont~ining primer extension unit; (k) steps (d) to (i) are 1~ pedted until detecting the detection moiety of the 30 primer e~ sion unit which is capable of base pairing with a nucleotide base 5' of the lepe~b region, said nucleotide base being the first nucleotide base of a type not incll~decl arnong the nucleotide bases in the core sequence of the tr mlcleo1ide repeats, dATP* in the given example; (1) the number of repeats as stated under O enables to calculate the number of trinucleotide repeats, CAG
3s in ~e given example, ~llel~;rol~e, enables the de~e~llunation of the exact lepelilion number.

- 21709Sl 1~
In order to better understand the preferred embodim~nt of the present invention, refe~ellce is made to Figure 2 which is a sch~m~hc depiction of modified and more efficient methods aimed at quantif~ing the number of CAG
trinucleo~tide le~)faLs in e~ .;..ed nucleic acid sequence which includes: (a) if s the n~lc.leic acid of illteleal is not already single stranded, treating a sample co..tA;~.;ng the nucleic acid to obtain unl)aired nucleotide bases sp~nnin~ the epeàls and fl~nking regions; (b) under hybridization conditions, cont~cting the ~paired nucleotide bases with an oligonucleotide primer, having a sequence which is complemPnt~ry to a stretch of nucleotide bases situated preferably 3' of o the reped~s region in the e~mined single strand nucleic acid template, l.reff,ably, the 3'-end of said oligonucleotide primer is annealed to the first nucleotide 3' of the repea~ region; (c) providing means to ensure that the eY~mined nucleic acid and the oligonucleotide primer are confined to a reaction chamber at all further steps; (d) the template primer hybrid is contacted with IS primer eytp-ncion units which are capable of base pairing with the first and second nucleotide bases in the core sequence of the repeats, dGTP and dTTP in the given example, and a template depPn~ent extension enzyme; alternatively (d~ the template pnmer hybrid is contacted with a primer extension unit which is capable of base paillng with the first nucleotide bases in the core sequence of 20 the repeals, dGTP in the given example, and a template dependent extension enzyme; (e) after a suitable incubation time non-incorporated primer extension units are elimin~te~l preferably washed away; (f) the template primer hybrid, now said primer elongated by two units, is contacted with a primer extension unit which is capable of base painng with the third nucleotide base in the core 2s sequence of the repeats, dCTP in the given example; a detection moiety col-~ai~ -g primer e~rtPnSion unit which is capable of base pairing with a nucleotide base 5' of the r~eals region, said nucleotide base being the first nucleotide base of a type not included among the nucleo~de bases in the core sequence of the trin-lcleotide r~peal~, dATP* in the given example; and a 30 template dependent e~ sion enzyme; alternatively (~) the template prirner hybrid, now said primer elongated by one unit, is contacted with pnmer Ç~f~cion units which are capable of base pairing with the second and third nucleotide bases in the core sequence of the .epeals, dTTP and dCTP in the given example; a detection moiety co..~ primer extension unit which is 3s capable of base ~ g with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not inchlde~ among thenucleotide bases in the core sequence of the trinucleotide lepeals, dATP* in thegiven eYP~nple; and a template llepen~ent eAI~-.sion enzyme; (g) after â suitable ~_ 18 incllb~tion time non-inco1po1ated primer extension units are elimin~te~l pfefe1ably washed away; (h) detecting for the presence of the detection moiety co~ . primer cYt~nsion unit; (i) steps (d) to (h) are rel~eA~ until detecting the detection moiety of the primer eYt~nsion unit which is capable of base s pairing with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not included arnong the nucleotide bases in the core sequence of the trinucleotide re~eals, dATP* in the given example; (i) the number of 1e~eals as stated under (i) enables to calculate the number of trinucleotide repeats, CAG in the given example, therefore, enables o the ~let~ ~ .. .i..~*on of the exact repclilion number.

The human genome contains two alleles of each gene. Each of the alleles is located on one of the chromosomes m~king up a pair of homologue chromosomes. Alleles characterized by triple repeats are very polymorphic by lS nature, therefore most individuals contain non-identical alleles for each of their trinucleotide repeats contAinin,~ genes, said alleles differ from one another bythe number of trinucleotide repeats they contain. As explained, for diagnostic and prognostic pu~.~oses, it is important and crucial to dete11nille the exact number of tnnucleotide repeats cont~in~o~3 in both alleles of the exarnined gene.
20 In order to better understand the prefe11ed embodiment of the present invention, r~fere11ce is made to Figure 3 which is a schematic depiction of a method aimed at siml)lt~neous quantification of the number of a CAG trinucleotide repeats in two alleles each contains a different number of said repeats. According to this embodiment, steps of incorporating pTimer extension units complementary to 2s the core sequence of the repeats are performed as detailed above until a detection of the detection moiety cont~ining~ primer extension unit which is capable of base pairing with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not included arnong the nucleotide bases in the core sequence of the trinucleotide repeats, is made, and30 the m~itude of the signal recorded. Counting these steps enables to determine~e number of trinllcleotide repeats in the allele cont~ining the lower number ofel~eals. After records have been made, steps of incorporating primer extension units co~nplçmentAry to the core sequence of the repeats are continue~ as detailed until fuIther detection of the detection moiety co~tAin;np~ primer 3s e~t~ nsion unit which is capable of base pairing with a nucleotide base 5' of the r~peats region, said nucleotide base being the first nucleo~de base of a type not inr,~ le~ among the nucleotide bases in the core sequence of the trinucleotide 1~,~ts. Co~ting these steps and adding the reslllte~ number to the former . 2170951 . ~ ,9 enables to ~ete~...;..e the number of trinucleotide repeats in the allele con~ gthe greater number of repeats.

Under this desc~ ion, the ~etection moiety of the primer e~t~ncion unit s is non-removable, thus, as the second signal is obtained a s~n~ g of signals is made. E~refelably a removable ~etection moiety may be used and removed after letectin~ the signal from the allele cont~ p less ~pedtS, ~ltçrn~tively~ a di~e.ent kind of detection moiety may be used, hence, the detection of the second signal is simpler since in both cases the second detection, as the first, is o based on all or non-detection.

The embo~ime~ts of the present invention are also useful for dete~ g the nllmber of head to tail repeat sequences of two or more nucleotide bases, provided that the core sequence consists of no more than three types of lS nucleotide bases.

Methods according to l~lefe.led embodiments of the present invention enjoy a nnmber of advantages relative to the prior art:

First, these are high resolution methods capable of a precise and unambiguous det~,...i.~tion of the number of trinucleotide repeats in a selectedlocus contained in a genetic material sample and is therefore suitable for monitoring small variations in trinucleotide repeat nurnbers.

2s Second, the methods according to preferred embodiments of the present invention is c~p~ble of rlictin~lichin~ between size variations due to trim)cleotide lelpedls e~rA~siorl/de expansion or other molecular events such as~e loss or formation of a restriction enzymes recognition site, or such as the loss or gain of sequences due to deletions or insertions.
Third, these methods do not include any kind of gel electrophoresis or other size based separations and therefore may be easily ~ntom~ted diminichin~
the r~ e~,ent for highly skilled personnel for their routine execution.

Four~, ordir~y persormel are sufficient for intel~lcling the results and for calclll~tin~ the trinucleotide sepeals number.

.- 2170951 Finally, the high GC contellt of some of the trinucleotide repeats create gel rnigration artifacts due to the formation of strong secondary structures. Since ~e afo~-ne~l;oned methods of the invention aimed at the de~ hon of trinucleotide repeals number are gel electrophoresis indepen~ent these ar~facts,s attributed to gel electrophoresis dependent methods, are cilcu~l-rented.

The above described embo-liments of the present invention may be made more efficient in term of costs by reusing materials, that is, after each step of the ones described, re~nts are collected and are reused in further steps. A re-o concentration and purification procedures may be needed before reuse of thesereaction reagents is made.

The specific application of the inventive method for the quantification of trinucleotide repeals in genes known to carry such repeats is presently a IS pl~;felled embodiment. In this application the method may be used as a diagnostic assay to determine the number of trinucleotide repeats present in individuals su~el~lg from, or showing syrnptoms of, diseases known to be caused by eXI~nsion of such repeats in or close to specific genes. The method may also be applied for simultaneous screening of apparently healthy 20 individuals to dele~ e whether any of them are carriers of any such repeats.
This is the case, for example, in the well elucidated Hnntin~on's disease in which lice~ce~1 individuals have e~cp~n~e~ tnnucleotide repeats in at least oneallele encoding ~e IT15 gene. Furdlermore, the method may also be applied for scf~enil~g embryos by analyzing sarnples of arnniotic fluid cells to detç~ ;ne 2s whether the embryos have any known trinucleotide repeats expansion in one or two or none of the alleles encoding genes known to be involved in specific genetic diseases in which such expansions are involved.

The genetic material to be analyzed may, in principle, be any RNA or 30 DNA obt~ ed from the tissues or body fluids of hllm~ns ~nim~lc or plants or obtained ~om cultures of microor~nicmc or hllm~n animal or plant cells or nucleic acid synthesi7e~l by çYtçnsion enzyme. The genetic material may ~h~ tively be obtained from non-living sources suspected of con~ ng matter from living or~ ,-.. sources, as may be the case when applying the method in 3s forensic medicine for de~clil,g and identifying specific nucleotide sequencesese~t in or on samples of clo~ing, Çu~n.lure, weapons and other items found at the scene of a crime. In this instance, the genetic material obtained is usuatly - 21709~1 in the form of DNA, since any RNA in such samples would normally have been degraded by ribonucleases.

The sample of nucleic acids can be drawn from any source and may be s natural or synthetic. The sample of nucleic acids may be made up of deoxyribonucleic acids, ribonucleic acids, or copolymers of deoxyribonucleic acid and ribonucleic acid. The nucleic acid of i~t~esl can be synthesi7e~
enzymatically in vitro, or synthPsi7e-l non-enymatically. The sample col-t~ the nucleic acid or acids of in~ere~l can also comprise extr~g~nomic 0 DNA from an or~nism RNA tr~nscripts thereof, or cDNA p~epared from RNA
s~ ts thereo Also, the nucleic acid of i~e~sl can be synthesi7e~ by ~e polymerase chain reaction.

The e~..i..ed nucleic acid can be made single stranded by using lS appro~,liate ~en~t lring conditions, which may include heat, aLkali, formamide, urea, glyoxal, enzymes, and combinations thereof.

Ex~min~*on of nucleic acids obtained from two or more individuals can be made simlllt~neously, for initial screening purposes. Furthermore, some 20 genes co.~t~ trinucleotide repeats which share identical repeat core sequence, nevertheless the trinucleotide repeats fl~nking regions differ substantially in their nucleotide base sequences. Therefore it is also possible to quantify the number of trinucleotide repeats in two or more genetic loci simultaneously.

2s The oligonucleotide primers can be any length or sequence, can be DNA
or RNA, or any modification thereof. It is necess~ry, however, that the length and sequence of the oligonucleotide ~)lilllels be chosen to op~mize the specificity of the hybridization to the target sequences of ll~tere~l.

Time and expense considerations tend to shift preferellce toward shorter oligonucleotides which are still sufficiently long to ensure high sequence specificity while at the same time en~-mng rapid, easy and acculate l,lepa,al on.

The oligomlcleotide primer may be any suitable species, preferably an 3s oligodeoxyribonucleotide, an oligoribonucleotide, a p~oteil- nucleic acid or a copolymer of deoxyribonucleotides, p~otelll nucleic acids and ribonucleotides.
The olipom~cleo~de primer can be either natural or synthetic. The - 2170~51 ~ 22 oligonucleotide primer can be synthesized enzymatically in vivo, enzymatically in vitro, or non-en_ymatically in vi~ro.

In addition, the oligonucleotide primer must be capable of hybridiing or S ~nne~ling with a stretch of nucleotide bases present in the nucleic acid of u~terc~l preferably 3' of the trinucleotide repeats region to be quantified. Oneway to accomplish the desired hybridization is to have the template dependçnt primer be sul~ lly complementary or fully complem~nt~ry to the known nucleotide bases sequence preferably 3' of the trinucleotide repeats region to be 10 q1~ntified. For convenience, in some in~t~nces the 3'-end of the oligonucleotide cr may overlap part of the repeat sequences. Furthennore, the oligonucleotide primer may be suitable for annealing with any of the strands in the e~-..;..ed nucleic acid and the types of primer çxtçn~ion units used changedaccordi~gly.
The single stranded examined nucleic acid and the oligonucleotide primer should be confined to a reaction chamber throughout the experimerlt~l steps in the method of the invention. This could be achieved, for example, by immobili7inP any of the mentioned molecules to a solid support. The 20 imrnobilization may be effected by binding the molecules to the solid supportvia (1) multiple ionic interactions between any of said molecules and the solid support; (2) multiple covalent bindings between the molecules and the solid ~u~oll, (3) direct single point coupling of the molecules to the solid support (4) indirect single point coupling of the molecules to the solid support via an 25 anchoring moiety conjugated to the molecules and a complement anchonng sites athched to the solid S~ppOll, e.g., biotin conjugated to the single stranded eY~ e-l nucleic acid or ~ltern~hvely the oligonucleotide primer or both and avidin, sLIeplavidin or antibiotin antibody are attached to the solid support; or magnetic beads are conjugated to the single stranded e~mined nucleic acid or 30 ~ sl;~tely the oligonucleotide pn~ner or both and the solid support is a magret or elec~.ol..agnet. (5) indirect multiple points coupling of the molecules to the solid support via anchoring moieties conjugated to the molecules at multiple sites and a complement anchoring sites ~ ed to the solid support. A
single point coupling of the e~c~mined nucleic acid, or ~ltenl~tively~ the 3s oligor.l~cle ~ide primer or both to the solid support, whether direct as under (3);
or indirect as under (4), is the ~lefe,.ed methodology since it ma~imi7es the accessibility of other reaction components to these molecules and form less steric constrains.

di~ on, the confinement of both the examined nucleic acid and the oligonucleotide primer to the reaction chamber may be effected by e~ll~pil1g these molecules using a porous membrane with a molecular weight cut off that 5 will facilitate the elimin~tion of primer ext~n~ion units via sepa~a~i~e filtration and will, at the same time, retain the above mentioned molecules. While by immobilizing the mentioned molecules to a solid support the template depPn~ent eYt~ncion enzyme is discarded along with the pnmer extencion units and therefore fresh enzyme should be added after each step, the enzyme, due to 10 its high molecular weight, is retained upon e,lllappi~lg the molecules using a porous membrane with a molecular weight cut off that will facilitate the el i~ ;on of primer extension units via separative filtration.

Any suitable extension moiety of the primer extension units may be used.
5 The extension moiety however should contain a 3'-OH group enabling further elongation of the oligonucleotide primer. The extension moiety of the primer e~t~nCion units may be deoxyribonucleotides, ribonucleotides or their 3'-OH
co..tA;n;.-~ analogs. Preferably, the extension moiety is a deoxyribonucleotide,such as dATP, dCTP, dGTP, dTTP and dUTP.
The elimin~tion of non-incorporated primer extension units from the reaction chamber, prece~lin~ further incorporation steps as delineated above, may be effected via physically removing said primer extension units from the reaction chamber by, for example, filtration as mentioned, or alternatively, by 25 destroying said primer extension units chernically or enzyrnatically wi~in the reaction chamber, for example by aLkaline phosphatase that will dephosphorilate said primer extension units, rendering them inapplicable for enzyrnatic incorporation.

Different versions of the methods for ~el~.. i"inp the number of trinucleotide repeats in a nucleic acid of interest are possible. In one version, the t~-nlpl~te is deoxyribonucleic acid, the primer is an oligodeoxyribonucleotide, oligoribonucleotide, protein nucleic acid, or a copolymer of deox~Tibomlcleotides, l~lole~n nucleic acids and ribonucleotides, and the 3s templ~t~ de~çn~Pnt enzyme is a DNA extension enzyme.

In a second version, ~e template is a ribonucleic acid, the primer is an olip~eox~ribon-l~leo1ide, oligoribonucleotide, p,oteill nucleic acid or a 2170g51 ~_ 24 copolymer of deo~yribonucleotides, protein nucleic acids and ribonucleotides, and the template dependent enzyme is a reverse transcriptase.

In a third version, the template is a deoxyribonucleic acid, the primer is s an oligoribonucleotide, and the enzyme is an RNA extension enzyme.

In a fourth version, the template is a ribonucleic acid, the primer is an oligoribonucleotide, and the template dependent enzyme is an RNA replicase.

The template dependent enzyme prefe.ably is confined to the reaction charnber. This could be achieved as described above by using a porous membrane with a molecular weight cut off that upon filtration will enable the elimin~tion of primer extension units but will retain the enzyme. Alternatively,the enzyrne may be linked via a long and flexible linking chain to a solid lS support, said solid support used also for immobilization of the exarnined nucleic acid and/or the oligonucleotide primer. The linking chain, being long and flexible, will allow the collision of the enzyme with its substrates, a prerequisite for catalysis. In both cases the addition of costly fresh enzyrne in every incorporation step, as described above, is elimin~te~l The nucleic acid of i,lte,est may contain non-natural nucleotide analogs such as deoxyinosine or 7 deaza 2' deoxyguanosine. These analogs destabilize DNA duplexes and could allow a primer annealing and extension reaction to occur in a double stranded sample without completely sep~aling the strands.
2s Any suitable detection moiety of the primer extension unit which is capable of base pairing with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not included arnong the nucleotide bases in the core sequence of the trinucleotide repeats, may be used.30 FullL~ ore, the detection moiety of the prirner extension unit should have physical and chemical prop~lies which do not interfere with its er~natic addition to the 3'-OH group of the elongated oligonucleotide p~irner. The ~letection moiety of ~e primer extension unit may facilitate the direct or indirect detection of its presence. For indirect detection the detection moiety of the 3s primer ~(f ~c;on unit may include a molecule of a type selected from the group concictin~ of enymes, catalysts, haptens, antibodies, sub~llatcs, coenzymes and ch~nil~....;.~scence. ~efe.dbly the ~etection moiety of the primer extension . 2170951 unit f~cili~t~ the direct detection and may include a molecule of a type selected from the group consisting of fluorescence and radioactivity.

The detection moiety may be conjugated at any position to the primer s eYtension unit. Furthermore, the detection moiety may be of a kind that is rernovable or destructible by chemical, physical or enzymatic l-nanipulation.
Removable or distractible detection moieties of the primer extencinn unit which is capable of base pairing with a nucleotide base 5' of the repeats region, saidnucleotide base being ~he first nucleotide base of a type not included among the10 nucleotide bases in the core sequence of the trinucleotide repeats are preferably used, for reasons explained above, when the simultaneous quantification of two alleles each co.~t~;l.;..g a different number of tnnucleotide repeats is desired.

The detection of the detection moiety, to a very large extent, is lS dependent upon its chemical and physical properties. Any suitable detection approach may be selected. In a case where the detection moiety of the primer extension unit is non-removable, the detection, whether direct or indirect, is prefe.~dbly carried out in the reaction camber. On the other hand, in a case where the detection moie~ of the primer extension unit is removable, the 20 detection~ whether direct or indirect, may be carried out in a di~erent chamber.

The ongoing research to determine the genetic basis for diseases and the advent of technologies such as the Polymerase Chain Reaction (PCR) has resulted in the discovery and complete sequencing of so far seven genes in 25 which trinucleotide repeats expansion would lead to either no expression of the gene product or e~ ession of a product which is qualitatively or qu~llitalively impa~ed and thereby res~ltin,~ in a disease. Since trinucleotide repeats have been observed within or close to a number of human genes by gene bank se~ches, it is conceivable that trinucleotide amplification may be involved in 30 the ~lls~tion of other genetic ~lice~ses as well. There is thus an ever expanding field of application of the above method of the invention.

Since di- and trinucleotide repeats are highly polyrnorphic, that is for each gene characte.~d by such repeats at least few dozen alleles L~l~,g in 35 their lepedls nurnber exist, the method of the invention may also be applied in the field of forensic medicine in which polymoIphism in specific genes can be dete.~ ncd in, for e~a."plc, blood or semen samples obtained at the scene of a crime and the results used to indicate whether or not a particular sl~spect was ~_ 26 involved in the crime. Similarly, the aforesaid dete~ ation may also be used to dete~ e whether a certain male individual is the father in cases of disputed pA t~

s There is evidence that cerhin cancers may be the result of di- and trinucleotide-eYpAncion in many gene targets, therefore, the present invention may be used as a cancer early diagnostic and prognostic tool.

Another application of the present methods is the detection of microor~AnicmC in a sample on the basis of the presence of specific sequences in the sample. For eYAmrle, an individual suspected of being infected by a rnicroorgAnicm such as a bacteria or virus, can be tested by using an oligonucleotide primer which anneal only with a specific bacterial and/or viral DNA sequences and not with sequences present in the examined individual. The oligonucleotide primer's sequence is selected to enable its hybridization 3' of a stretch of nucleotide bases composed of one to three types of nucleotide bases followed by a di~lent type of nucleotide base in the examined sequence and a procedure similar to the ones described above is carried out. Detecting the detection moiety of the primer extension unit that is complementary to said di~re,lt type of nucleotide base is an indication of the presence of the c~A~n;~.ed genetic material in the sample. One example of such an application isin the screening of individuals for the presence of the AIDS virus.

The invention will now be further illustrated by the following examples:
EXAMPLES

Oligonucleotide primers and suitable detection moiety containing primer e~tension units aimed at the Quantification of trinucleotide repeats in the seven genes known to carry said repeats As mentioned, so far seven genetic ~iceAses each involving a unique genetic locus have been irnplicated with trinucleotide repeat mutations. These include: Fragile XA (A site, Martin Bell) syndrome (FRAXA); Kennedy ~i~e~ce 3s (spinal and bulbar muscular allOp~ly, SBMA); Myotonic dystrophy (Cu~sckn~nn Steiner~ DM); ~lmtin~Qn'S disease (HD); Spinoce~bellar ataxia ~pe 1 (SCAl); Fragile XE (E site) mental retardation (FRAXE MR); and Dc~ olublal paUidoluysian alloph~r (DRPLA).

- 21709Sl Table I lists six of these ~ise~ses which are known to result from ~inucleotide ~epe~ls e~pallsion; ~ropliate 18 mer oligonucleotide primers, the upper primer being suitable for hyblidization with a (-) strand therefore is s suitable for hybridization with the (-) strand of single stranded DNA, whereasthe lower primer being suitable for hybridization wi~h a (+) strand therefore issuitable for hybri~li7~tion with the (+) st~and of single stranded DNA or enzymatically ~anscribed RNA; and detection moiety cont~inin~ pIimer e~lel-cion units suitable for del~ .,.;.-;--~ ~he number of trinucleo~de repeats as o described above.

Table I:

Disease and Primers: Extension (repeats core units:
sequence):

Fragile XA 5' AGGGGGCGTGCGGCAGCG 3' dT/ATP
(CGG)n 5' CGGGCGCTCGAGGCCCAG 3' dT/ATP
Kennedy disease 5' GCCAGTTTGCTGCTGCTG 3' dTTP
(CAG)n 5' CCTGGGGCTAGTCTCTTG 3' dATP

Myotonic dystrophy 5' GTCCTTGTAGCCGGGAAT 3' dATP
(GCT)n 5'ATGGTCTCTCATCCCCCC3' dTTP

Huntington's disease 5' GAGTCCCTCAAGTCCTTC 3' dTTP
(CAG)~, 5'CGGCGGTGGCGGCTGTTG3' dATP

Spinocerebellar 5' CCGGGACACAAGGCTGAG 3' dTTP
ataxia ~pe 1 5' CTGCTGCTGGATGCTGATG 3' dATP
(CAG)n D~llL~tolu~lal 5' CACCACCAGCAACAGCAA 3' dTTP
3s pallidoluysian 5' CCCAGAGTTTCCGTGATG 3' dATP
dt~ h,,t (CAG)n 2170~51 ~_ 28 Methods aimed at the quantification of trinucleotide repeats s Figure 1 illustrates a method aimed at quantifying the number of CAG
trinucleotide repeats in e~c~mined nucleic acid sequence containing 3 said repeats followed by a thyrnine residue (---CAG CAG CAG T---). The sample co.-~ in~. the nucleic acid of illte.esL is treated to obtain unpaired nucleotide bases sp~ the repeats and fl~nkin~ regions (a). An oligonucleotide primer o is annealed to the nucleic acid template under hybridization conditions, preferably the 3'-end of said oligonucleotide primer is annealed to the first nucleotide 3' of the repeats region (b). The exarnined nucleic acid is attached to a solid support (referred to as "S" in the Figure) indirectly via a single pointcoupling consicted of anchoring moiety conjugated to the molecules and a complement anchoring site attached to the solid support (c). l~e template prirner hybrid is contacted with a dGTP primer exte~sion unit which is capable of base pairing with the first nucleotide base in the core sequence of the repeats, and a template dependent extension enzyme, referred to as "E" in the Figure (d).After a suitable incubation time, non-incorporated dGTP primer extension unit is washed away along with the extension enzyme (e). The template primer hybrid, now said primer elongated by one guanine residue, is contacted with dTTP primer extension unit which is capable of base pairing with the second nucleotide base in the core sequence of the repeats, and a template dependent eAlel.sion enzyme (f). After a suitable incubation time non-incorporated dTTP
primer eYt~ncion units are washed away (g). The template primer hybrid, now said prirner elongated by guanine followed by thirnine residues, is contacted wi~ dCTP primer extension unit which is capable of base pairing with the third nucleotide base in the core sequence of the repeats, a dATP detection moiety col-t~ primer extension ur~it, efe~led to as "*" in the Figure, which is capable of base pairing with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not included arnong the nucleotide bases in the core sequence of the ~inucleotide repeats, and a templ~te depend~nt extension enzyme O. after a suitable incubation tirne, non-inc~ oraled dCTP and dATP* primer eYtencion units are washed away (i).
3~ Det~c~ for the presence of the detection moiety (*) co~ dATP primer c~ ;on unit (j). Steps (d) to (j) are repeated altogether dlree tirnes until ~et~ the detection moiety (*) of the dATP primer e~tension unit (j3). The 2170~51 ~_ 29 number of repeats equals the nurnber of the CAG trinucleotide repeaes, thereforeenabling ~etemtin~thon of the exact repetition number.

s Figure 2 illustrates a modified and more efficient method aimed at .lu~~ ing the number of CAG trinucleotide re~eals in ex~tmined nucleic acid sequence co..~ 3 said repeats followed by a thimine residue (---CAG CAG
CAG T---). The sample contStining the nucleic acid of il~teles~ is treated to o obtain ullpaired nucleotide bases sp~.-n;..~ the repeats and fl~tnkin_ regions (a).
An oligonucleotide primer is annealed to the nucleic acid terr,plate under hybridization conditions, preferably the 3'-end of said oligonucleotide primer is stnnestled to the first nucleotide 3' of the repeats region (b). The examined nucleic acid is attached to a solid support (S) indirectly via a single point lS coupling conci~te~l of anchoring moiety conjugated to the molecules and a comple~e~t anchoring site attached to the solid support ~c). The template primer hybrid is contacted with dGTP and dTTP primer extension units which are capable of base pstiring with the first and second nucleotide bases in the core sequence of the repeats and a template dependent extension enzyme (E) (d). The 20 template primer hybrid, now said primer elong~ted by a guanine and a thimine residues, is contacted with a dCTP primer extension lmit which is capable of base p~tirin~ with the third nucleotide base in the core sequence of the repeats, a dATP detection moiety cont~tinin_ prirner extension unit (*) which is capable of base pairing with a nucleotide base 5' of the repeats region, said nucleotide2s base being the first nucleotide base of a type not included among the nucleotide bases in ~e core sequence of the trinucleotide repeats, and a template depe,ldellt e~l~nsion enzyme (f). After a suitable incubation time non-inco~ ated dCTP and dATP* primer extencion units are washed away (g).
Dete~!;np for the presence of the dATP* detection moiety contStining~ primer 30 ~ .c;on unit (h). Steps (d) to (h) are repeated altogether three times until-~ehc~ g the detection moiety (*) of the dATP primer extension unit (h3). The mlmb~r of re~ed~ equals the number of the CAG trinucleotide repeats, therefore enabling ~et~ sttion of the exact repetition number.

3s EXAIVIPLE 3 Figure 3 illuallales a metho-l aimed at simnltstneous qu~ltirlcation of the n~lmb~ of CAG trinucleotide le~,cats in two alleles the first co.~tSt;~ g one and ~_ 30 2170~51 the secon~l two said repeals followed in both cases by a thimine residue. Steps of incorporating pli ~c~ eYtPncion units complementary to the core sequence of the repeats are ~e ro-...ecl as detailed above until a ~letection of the detection moiety cor.~ primer extension unit (A*) which is capable of base pairing s with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the tTinucleotide repeats, is made and the magl~itude of the signal recorded (*). Counting these steps enables to ~lete~ ;..e the number of trinucleotide repeats in the allele co~ g the lower number of repeats. After 0 records have been made, steps of incorporating primer eYtension units complementary to the core sequence of the repeats are cont;nl~e~ as detailed until further detection of the detection moiety co..~ g primer extension unit (A*) which is capable of base pairing with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not IS included among the nucleotide bases in the core sequence of the tnnucleohde repeats is made and the ma~itl~de of the additive signal ($*) or a di~erent signal, denoted as a closed box in the Figure, is recorded. Counting these steps(1 step in the given example) and adding the resulted number to the folmer (1 step in the given example) enables to ~ete~ ;..e the number of trinucleotide 20 repeats in the allele cont~ining the greater number of repeats (2 in the given example).

A diagnostic kit for quantifying trinucleotide re~eat mutahons A dia~ostic kit for carIying out a preferred embodiment of ~e methods rdil~g to the present invention detailed above may contain ~e following CO--c! ;l ~)entc a) any number of suitable oligonucleotide prirners;
b) one to three ~ nc~ extension units;

3s c) Fur~er one or more primer extension units of a type not included under b), said primer ~ .sion units co.~ p a detection moiety;

d) a suitable template depPn~ent e~tencion enzyrne for car~ing out the primer eAlension unit incorporation, or extension, steps of the method;

e) suitable buffer/s in aqueous solution for carrying out the ~nne~lin~
s ext~nsion, wash and detection steps of the method; and When the kit is to be used for Fragile XA; Kennedy disease; Myotonic d~.LIophy; ~l~ntin~on's disease; Spinocerebellar ataxia type l; Fragile XE
m~nt~l retardation; and De.llalol,lbral pallidoluysian atrophy, it may contain, o for example, any one or all of the specific oligonucleotide primers listed in Tables I above for quantifying the tnnucleotide repeats eYr~nsions occurring in these genes. When the kit is to be used in the screening for the presence of one or all of the various listed genetic ~ise~ses it may contain any suitable number of the oligonucleotide primers in any suitable combination. Different lS combinations of primers may be included in kits for different intended populations. When the kit is to be used forensic or paternity typing it may contaul any combination of specific oligonucleotide ~ els, each designed to quanlif~ a particular trinucleotide repeats contained in any of the mentioned orother genes. Depending on the circumstances, all of the kits may also contain 20 any nl-mber of additional oligonucleotide primers suitable for determinin~. the presence or absence of a DNA sequence corresponding specifically to the presence of a pathogen, for example, the presence of the AIDS virus.
Accordu~gly, one kit may be used for testing any number of genes or gene, and this only requires that the kit contain a number of the specific oligonucleotide2s primers, all the other components of the kit being the same in all cases.

While the invention has been described with respect to a limited nllmbPr of embodiments, it will be appreciated that many variations, modifications and other applications of the invention may be made.
An ulsllul~,cnt for q,lalllifying trinucleotide repeat mutations EXAMPLE S

3s Figure 4 i~ atcs an aulo-~ate~ instrument suitable for eYecllhnp. thesteps in the method of the present invention for the ~let~ ...;n~1;on of the number of trinllcl~olide r~pea~ co~ led in specific genetic loci. The steps of the reaction are carried out in â reaction chamber (1) to which reagents may be 2170~51 added or elimin~te~ via controllable valved tubing. Within the reaction chamber included is a solid support (2) suitable for immobilizing the nucleic acid of ~l~resl, the oligonucleotide primer or both. ~ltern~tively or additionally the reaction chamber outlet cont~ins a porous membrane (3) facilit~ting the s retention of at least the nucleic acid of illtere~L and the oligonucleotide primer while facilit~tin~ the sepdlali.re filtration of primer extension units. Delector device (4) aimed at the detection of the detection moiety contained by the prima e~cten~ion unit which is capable of base p~irin~ with a nucleotide base S'of the rcpe~ region, said nucleotide base being the first nucleotide base of a o type not included among the nucleotide bases in the core sequence of the trinucleotide repeats is located at the reaction chamber in cases where said detection moiety is of an non-removable type, alternatively, the detector device(5) may be located elsewhere, for exarnple at the oudet, in cases where said detection moiety is of a removable type. The reaction chamber further contains lS two to three (6) inlets each connected to a reservoir (7) collt~inin~ one to two types of the primer extension units capable of base pairing with the first (lst), second (2nd) and third (3rd) nucleotide base in ~e core sequence of the tnnucleotide repeats. The second or ~ird of said reservoir, depending upon the specific application, further contains a detection moiety cont~ining primer 20 ~Ytension unit (1:)) which is capable of base painng with a nucleotide base 5' of the repeats region, said nucleotide base being the first nucleotide base of a type not included among ~e nucleotide bases in the core sequence of the trinucleotide repeats. Further reservoirs and inlets for the a.lministration of mater als such as a suitable template dependent enzyme; reaction buffer, wash 2s bu~er/s, detection buffer/s (sensitizer) and ~e like materials may also be included (8). The reaction chamber and any reversoire may be temperature controlled (9). Tubing connecting the reaction chamber oudet with any of dle reservoirs may be added as well as a reconcentration (10) and/or purification (11) device aimed at ~e reuse of discarded materials. The instrument may be 30 G~ ated m~n~ y or preferably automatically for example the instrument o~c~alion may be controlled by a built in or external computer.

Claims

1. A method of quantifying the number of trinucleotide repeats in an examined nucleic acid sequence, comprising the steps of:
(a) if such nucleic acid is double stranded, treating the sample containing the nucleic acid of interest to obtain unpaired nucleotide bases spanning the trinucleotide repeats and their flanking regions, or, if the nucleic acid of interest is single stranded, directly employing step (b);
(b) contacting the unpaired nucleotide bases spanning the number of trinucleotide repeats and their flanking regions with an oligonucleotide primer for hybridizing with a stretch of nucleotide bases present in the nucleic acid of interest partially or fully 3' of the nucleotide repeats to be quantified, so as to form a duplex between the primer and the nucleic acid of interest;
(c) providing means for confining the examined nucleic acid and the oligonucleotide primer to a reaction chamber at all further steps;
and further comprising the cycled steps of:
(d) contacting the template primer duplex with a first primer extension unit for base pairing with one of the nucleotide bases, in the core sequence of the trinucleotide repeats, and with a template dependent extension enzyme;
(e) eliminating non-incorporated units of said first primer extension units;
(f) contacting the template primer duplex, which primer is now extended by one unit as described in step (d), with a second primer extension unit for base pairing with a second nucleotide base, in the core sequence of the repeat, said second nucleotide base being located adjacent to and immediately 5' of the nucleotide base employed under step (d), and with a template dependent extension enzyme;
(g) eliminating non-incorporated units of said second primer extension units;
(h) contacting the template primer duplex, which primer is now elongated by one further additional unit as described in step (f), with:
(i) a third primer extension unit for base pairing with a third nucleotide base, in the core sequence of the repeat, said third nucleotide base being located adjacent to and immediately 5' of the nucleotide base under step (f);
(ii) a detection moiety which is conjugated with a fourth primer extension unit for base pairing with a nucleotide base 5' of the repeats, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the trinucleotide repeats, said detection moiety which is conjugated with said fourth primer extension unit may be present in selected cycles of this stage; and (iii) a template dependent extension enzyme;
(i) eliminating non-incorporated units of said third and fourth primer extension units;
(j) if step (h) included said detection moiety which is conjugated with said fourth primer extension unit, detecting the presence of said detection moiety; and if no detection is obtained, (k) repeating steps (d) to (j) until said detection moiety is detected, said detection of said detection moiety being indicative of the number of trinucleotide repeats included in the nucleic acid of interest.

2. A method as in claim 1, wherein the nucleic acid of interest is selected from the group consisting of synthetic and natural deoxyribonucleic acid, ribonucleic acid, and a copolymer of deoxyribonucleic acid and ribonucleic acid.

3. A method as in claim 1 wherein the oligonucleotide primer is selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, protein nucleic acids and copolymers of oligodeoxyribonucleotides, protein nucleic acids and oligoribonucleotides.

4. A method as in claim 1, wherein the oligonucleotide primer is substantially complementary to said complementary sequence.

5. A method as in claim 1, wherein the oligonucleotide primer is fully complementary to said complementary sequence.

6. A method as in claim 1, wherein the confining of the nucleic acid and the oligonucleotide primer to a reaction chamber at all steps is effected by a process selected from the group of techniques consisting of direct and indirect-, single and multiple- immobilization to a solid support, combinations thereof and molecular weight cut off filtration.

7. A method as in claim 1, wherein the extension moiety of said first second and third primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides and their 3'-OH
containing analogs, and said fourth primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides, dideoxynucleotides and their analogs.

8. A method as in claim 1, wherein the extension moiety of said first second and third primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP and their 3'-OH containing analogs and the extension moiety of said fourth primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP, ddATP, ddCTP, ddGTP, ddTTP and their analogs.

9. A method as in claim 1, wherein the elimination of the primer extension units is effected by a process selected from the group of techniques consisting of washing. filtering and chemical, enzymatic and physical destruction.

10. A method as in claim 1, wherein the detection moiety of the primer extension unit is situated at any position on said primer extension unit and is selected from the group of types consisting of direct and indirect detection moieties.

11. A method as in claim 1, wherein the detection moiety of the primer extension unit is selected from the group of types consisting of removable, non-removable and destructible chemical groups.

12. A method of quantifying the number of dinucleotide repeats in an examined nucleic acid sequence, comprising the steps of:
(a) if such nucleic acid is double stranded, treating the sample containing the nucleic acid of interest to obtain unpaired nucleotide bases spanning the dinucleotide repeats and their flanking regions, or, if the nucleic acid of interest is single stranded, directly employing step (b);
(b) contacting the unpaired nucleotide bases spanning the number of dinucleotide repeats and their flanking regions with an oligonucleotide primer for hybridizing with a stretch of nucleotide bases present in the nucleic acid of interest partially or fully 3' of the nucleotide repeats to be quantified, so as to form a duplex between the primer and the nucleic acid of interest;
(c) providing means for confining the examined nucleic acid and the oligonucleotide primer to a reaction chamber at all further steps;
and further comprising the cycled steps of:
(d) contacting the template primer duplex with a first primer extension unit for base pairing with one of the nucleotide bases, in the core sequence of the dinucleotide repeats, and with a template dependent extension enzyme;
(e) eliminating non-incorporated units of said first primer extension units;
(f) contacting the template primer duplex, which primer is now extended by one unit as described in step (d), with:
(i) a second primer extension unit for base pairing with a second nucleotide base, in the core sequence of the repeat, said second nucleotide base being located adjacent to and immediately 5' of the nucleotide base under step (d);
(ii) a detection moiety which is conjugated with a third primer extension unit for base pairing with a nucleotide base 5' of the repeats, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the dinucleotide repeats, said detection moiety which is conjugated with said third primer extension unit may be present in selected cycles of this stage; and (iii) a template dependent extension enzyme;
g) eliminating non-incorporated units of said second and third primer extension units;
(h) if step (f) included said detection moiety which is conjugated with said third primer extension unit, detecting the presence of said detection moiety; and if no detection is obtained, (i) repeating steps (d) to (h) until said detection moiety is detected, said detection of said detection moiety being indicative of the number of dinucleotide repeats included in the nucleic acid of interest.

13. A method as in claim 1, wherein steps (d) and (f) and steps (e) and (g) are combined under two successive single steps, in the first combined single step said template-primer duplex is contacted with the first and second primer extension units for base pairing with two adjacent nucleotide bases in the core sequence of the repeats, and a template dependent extension enzyme, while in the second combined single step said non-incorporated first and second primer extension units are eliminated.

14. A method as in claim 13, wherein the nucleic acid of interest is selected from the group consisting of synthetic and natural deoxyribonucleic acid, ribonucleic acid, and a copolymer of deoxyribonucleic acid and ribonucleic acid.

15. A method as in claim 13 wherein the oligonucleotide primer is selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, protein nucleic acids and copolymers of oligodeoxyribonucleotides, protein nucleic acids and oligoribonucleotides.

16. A method as in claim 13, wherein the oligonucleotide primer is substantially complementary to said complementary sequence.

17. A method as in claim 13, wherein the oligonucleotide primer is fully complementary to said complementary sequence.

18. A method as in claim 13, wherein the confining of the nucleic acid and the oligonucleotide primer to a reaction chamber at all steps is effected by a process selected from the group of techniques consisting of direct and indirect-, single and multiple- immobilization to a solid support, combinations thereof and molecular weight cut off filtration.

19. A method as in claim 13, wherein the extension moiety of said first second and third primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides and their 3'-OH
containing analogs, and said fourth primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides, dideoxynucleotides and their analogs.

20. A method as in claim 13, wherein the extension moiety of said first second and third primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP and their 3'-OH containing analogs and the extension moiety of said fourth primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP, ddATP, ddCTP, ddGTP, ddTTP and their analogs.

21. A method as in claim 13, wherein the elimination of the primer extension units is effected by a process selected from the group of techniques consisting of washing, filtering and chemical, enzymatic and physical destruction.

22. A method as in claim 13, wherein the detection moiety of the primer extension unit is situated at any position on said primer extension unit and is selected from the group of types consisting of direct and indirect detection moieties.

23. A method as in claim 13, wherein the detection moiety of the primer extension unit is selected from the group of types consisting of removable, non-removable and destructible chemical groups.

24. A method as in claim 1, wherein steps (f) and (h) and steps (g) and (i) are combined under two single steps, in the first combined single step said template-primer duplex, now said primer extended by one unit, is contacted with:
(i) said second and third primer extension units for base pairing with two adjacent nucleotide bases in the core sequence of the repeats;
(ii) a detection moiety which is conjugated with said fourth primer extension unit, said detection moiety which is conjugated with said fourth primer extension unit may be present in selected said cycles of this stage; and (iii) a template dependent extension enzyme;
while in the second combined single step, said non-incorporated second third and fourth primer extension units are eliminated.

25. A method as in claim 24, wherein the nucleic acid of interest is selected from the group consisting of synthetic and natural deoxyribonucleic acid, ribonucleic acid, and a copolymer of deoxyribonucleic acid and ribonucleic acid.

26. A method as in claim 24 wherein the oligonucleotide primer is selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, protein nucleic acids and copolymers of oligodeoxyribonucleotides, protein nucleic acids and oligoribonucleotides.

27. A method as in claim 24, wherein the oligonucleotide primer is substantially complementary to said complementary sequence.

28. A method as in claim 24, wherein the oligonucleotide primer is fully complementary to said complementary sequence.

29. A method as in claim 24, wherein the confining of the nucleic acid and the oligonucleotide primer to a reaction chamber at all steps is effected by a process selected from the group of techniques consisting of direct and indirect-, single and multiple- immobilization to a solid support, combinations thereof and molecular weight cut off filtration.

30. A method as in claim 24, wherein the extension moiety of said first second and third primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides and their 3'-OH
containing analogs, and said fourth primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides, dideoxynucleotides and their analogs.

31. A method as in claim 24, wherein the extension moiety of said first second and third primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP and their 3'-OH containing analogs and the extension moiety of said fourth primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP, ddATP, ddCTP, ddGTP, ddTTP and their analogs.

32. A method as in claim 24, wherein the elimination of the primer extension units is effected by a process selected from the group of techniques consisting of washing, filtering and chemical enzymatic and physical destruction.

33. A method as in claim 24, wherein the detection moiety of the primer extension unit is situated at any position on said primer extension unit and is selected from the group of types consisting of direct and indirect detection moieties.

34. A method as in claim 24, wherein the detection moiety of the primer extension unit is selected from the group of types consisting of removable, non-removable and destructible chemical groups.

35. A method aimed at simultaneous quantification of the number of nucleotide repeats in two alleles, each of said alleles containing any number of said repeats wherein steps of incorporating primer extension units complementary to the core sequence of the repeats are performed as detailed under claim 1, 12, 13 or 24 until a detection of said detection moiety incorporated onto said primer annealed to a nucleic acid associated with the allele containing a lower number of repeats is made, and steps of incorporating primer extension units complementary to the core sequence of the repeats are continued until a further detection of a detection moiety incorporated onto said primer annealed to the nucleic acid associated with the allele containing a higher number of repeats is made.

36. A method as in claim 35, wherein the nucleic acid of interest is selected from the group consisting of synthetic and natural deoxyribonucleic acid, ribonucleic acid, and a copolymer of deoxyribonucleic acid and ribonucleic acid.

37. A method as in claim 35 wherein the oligonucleotide primer is selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, protein nucleic acids and copolymers of oligodeoxyribonucleotides, protein nucleic acids and oligoribonucleotides.

38. A method as in claim 35, wherein the oligonucleotide primer is substantially complementary to said complementary sequence.

39. A method as in claim 35, wherein the oligonucleotide primer is fully complementary to said complementary sequence.

40. A method as in claim 35, wherein the confining of the nucleic acid and the oligonucleotide primer to a reaction chamber at all steps is effected by a process selected from the group of techniques consisting of direct and indirect-, single and multiple- immobilization to a solid support, combinations thereof and molecular weight cut off filtration.

41. A method as in claim 35, wherein the extension moiety of said first second and third primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides and their 3'-OH
containing analogs, and said fourth primer extension unit is selected from the group consisting of deoxyribonucleotides, ribonucleotides, dideoxynucleotides and their analogs.

42. A method as in claim 35, wherein the extension moiety of said first second and third primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP and their 3'-OH containing analogs and the extension moiety of said fourth primer extension units is selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, TTP, ddATP, ddCTP, ddGTP, ddTTP and their analogs.

43. A method as in claim 35, wherein the elimination of the primer extension units is effected by a process selected from the group of techniques consisting of washing, filtering and chemical, enzymatic and physical destruction.

44. A method as in claim 35, wherein the detection moiety of the primer extension unit is situated at any position on said primer extension unit and is selected from the group of types consisting of direct and indirect detection moieties.

45. A method as in claim 35, wherein the detection moiety of the primer extension unit is selected from the group of types consisting of removable, non-removable and destructible chemical groups.

46. A diagnostic kit for detecting the presence of specific nucleotide repeat sequences in nucleic acid samples, comprising:
(a) any number of oligonucleotide primers for hybridizing with stretches of nucleotide bases present in nucleic acids of interest 3' of a nucleotide repeat sequence to be quantified, so as to form duplexes between each of the primers and the nucleic acids of interest;
(b) one to three primer extension units;
(c) further one or more primer extension units of a type not included under (b), said primer extension units containing a detection moiety;
and (d) a template-dependent extension enzyme.

47. An instrument for detecting the presence of specific nucleotide repeat sequences in nucleic acid samples, comprising:
(a) a reaction chamber for confining at least an examined DNA
template containing a repeat sequence and an oligonucleotide primer for annealing 3' of said repeat sequence, to which reagents may be added and eliminated through inlets and outlets;
(b) means for confining at least the examined nucleic acid and oligonucleotide primer to said reaction chamber;
(c) at least two inlets each connected to a reservoir containing one or two types of the primer extension units for base pairing with the first, second and third nucleotide bases in the core sequence of the repeat, one of said reservoir further contains said detection moiety containing primer extension unit for base pairing with a nucleotide base 5' the repeat region, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the repeat; and (d) a detector for detecting said detection moiety contained by said primer extension unit for base pairing with a nucleotide base 5' of the repeat sequence region, said nucleotide base being the first nucleotide base of a type not included among the nucleotide bases in the core sequence of the repeat.