Miller |
August 13, 1996 |
Method for nucleotide sequence amplifica |
tion |
Abstract |
This invention relates to an improvement target nucleotide sequence by using an e osmolyte in the reaction mixture of an a found that the use of a glycine-based os bands in the amplification product allow nucleotide sequence. For example, detect sequence, indicative of Huntington's Dis of a glycine-based osmolyte. |
of the procedure for amplifying a ffective amount of a glycine-based mplification procedure. It has been molyte reduces the appearance of stutter ing for easier detection of the target ion of the target trinucleotide repeat ease, is made clearer with the use |
Inventors: Miller; Glenn A. (Hopkinton, |
MA) |
Assignee: Genzyme Corporation (Cambridge |
, MA) |
Appl. No.: 326432 |
Filed: October 18, 1994 |
Current U.S. Class: 435/91.2; 435/91.21; |
435/91.5; 536/22.1; 536/23.1 |
Intern'l Class: C12P 019/34; C07H 021/00 |
Field of Search: 435/91.2,91.21,91.5 536 |
/22.1,23.1 |
References Cited |
U.S. Patent Documents |
5192659, Mar. 1993, Simons |
538602, 4Jan., 1995, Kacian et al. |
Other References |
Han, Jian et al. (1994), "Over Represent and (CGG) repeats in the human genome", 9 1735-1740. |
ation of the disease associated (CAG) Nucleic Acids Research vol. 22, No. |
Stallings, Raymond L. (1994), "Distribut in Different Categories of Mammalian Gen Barron, Lilias, H. et al. (1994), "A sin rich sequence immediately 3' to the unst cDNA shows almost complete disequilibriu in the Scottish population" Human Molecu Koide, R. et al. (1994), "Unstable expan torubral-pallidoluysian atrophy (DRPLA)" |
ion of Trinucleotide Microsatellites omic . . . " Genomics, 21:116-121. gle allele from the polymorphic CCG able CAG trinucleotide in the IT15 m with Huntington's disease chromosomes lar Genetics vol. 3, No. 1 173-175. sion of CAG repeat in hereditary denta- , Nature Genetics, 6:9-13. |
Litt, Michael, et al. (1993), "Shadow Ba leotide Repeats: Some Causes and Cures", |
nds Seen When Typing Polymorphic Dinuc- Biotechniques, vol. 15:280-284. |
Snell, Russell, G., (1993), "Relationshi and phenotypic variation in Huntington's Knight, S. J. L. et al., "Trinucleotide ion of a CpG Island in FRAXE Mental Reta |
p between trinucleotide repeat expansion disease", Nature Genetics, 4:393-397. Repeat Amplification and Hypermethylat- rdation", Cell, 74:127-134. |
Orr, Harry, T. et al., (1993), "Expansio repeat in spinocerebellar ataxia type 1" |
n of an unstable trinucleotide CAG , Nature Genetics, 4:221-226. |
Riess, Olaf, et al., (1993), "Improved P repeats causing Huntington's disease", H 6, p. 637. |
CR conditions for the stretch of (CAG).sub.n uman Molecular Genetics, vol. 2, No. |
Goldberg, Paul Y. et al. (1993) "A PCR m cleotide repeat expansion in Huntington vol. 2, No. 6, 635-636. |
ethod for accurate assessment of trinu- Disease", Human Molecular Genetics, |
Valdes, John M. et al. (1993), "A simple of Huntinton's disease", Human Molecular The Huntington's Disease Collaborative R Containing a Trinucleotide Repeat That I Disease Chromsomes" Cell 72:971-983. |
non-radioactive method for diagnosis Genetics, vol. 2, No. 6, 633-634. esearch Group (1993), "A Novel Gene s Expanded and Unstable on Huntington's |
Hauge, X. Y. et al. (1993), "A study of typing dinucleotide repeat polymorphisms vol. 2, No. 4, 411-415. |
the orgin of `shadow bands` seen when by the PCR" Human Molecular Genetics, |
Rees, William, A. et al. (1993), "Betain ion Dependence of DNA Melting", Biochemi |
e Can Eliminate the Base Pair Composit- stry, 32:137-144. |
Aslanidis, Charalampos, et al. (1992), " dystrophy region and mapping of the puta Buxton, Jessica, et al. (1992), "Detecti specific to individuals with myotonic dy |
Cloning of the essential myotonic tive defect", Nature, 355:548-551. on of an unstable fragment of DNA strophy", Nature, 355:547-548. |
Harley, Helen, G. (1992), "Expansion of variation in myotionic dystrophy", Natur |
an unstable DNA region and pheno-typic e, 355:545-546. |
Fu, Y. H. et al. (1992), "An Unstable Tr Myotonic Muscular Dystrophy", Science, 2 |
iplet Repeat in a Gene Related to 55:1256-1258. |
Andrew, Susan E. (1993), "The relationsh length and clinical features Huntington La Spada, Albert R., et al. (1991), "And ked spinal and bulbar muscular atrophy", |
ip between trinucleotide (CAG) repeat disease", Nature Genetics, 4:398-403. rogen receptor gene mutations in X-lin- Nature, 352:77-79. |
Kremer, E. J., et al. (1991), "Mapping o to a Trinucleotide Repeat Sequence p(CCG Verkerk, Annemieke et al. (1991), "Ident a CGG Repeat Coincident with a Breakpoin Variation in Fragile X Syndrome", Cell 6 |
f DNA Instability at the Fragile X ).sub.n ", Science, 252:1711-1714. ification of a Gene (FMR-1) Containing t Cluster Region Exhibiting Length 5:905-914. |
Yu, S. M. et al. (1991), "Fragile X Geno Region of DNA", Science, 252:1179-1181. |
type Characterized by an Unstable |
Miller, S. A. (1988), "A simple salting human nucleated cells", vol. 16, No. 3, |
out procedure for extracting DNA from p. 1215. |
Hippel, Peter H. et al. (1972), "DNA-Pro ular Biology and Departments of Chemistr pp. 231-300. |
tein Interactions", Institute of Molec- y and Biology, U of Oregon, Eugene, |
Melchior, William, B. et al. (1973), "Al of dA-dT and dG-dC Base Pairs in DNA", P 2, pp. 298-302. |
teration of the Relative Stability roc.Nat. Acad. Sci. , vol. 70, No. |
Primary Examiner: Wityshyn; Michael G. |
Assistant Examiner: Prats; Francisco C. |
Attorney, Agent or Firm: Salcedo; F. Bra |
d |
Claims |
The invention claimed is: |
1. In the procedure for amplifying a tar trinucleotide repeats, the improvement c of a trimethylglycine to the reaction mi procedure wherein fewer amplification pr the target nucleotide sequence are produ absence of trimethylglycine. |
get nucleotide sequence containing omprising adding an effective amount xture of a Taq polymerase chain reaction oducts which do not correspond to ced than would be produced in the |
2. The procedure of claim 1 wherein the is indicative of a disease state. |
nucleotide sequence being amplified |
3. The procedure of claim 1 wherein the 4. A kit for amplifying a target nucleot repeats, comprising in separate containe |
target nucleotide sequence is a DNA. ide sequence containing trinucleotide rs: |
a) the components for a Taq polymerase c |
hain reaction; and |
b) a trimethylglycine. |
5. The kit of claim 4 wherein the the nu is indicative of a disease state. |
cleotide sequence being amplified |
6. The kit of claim 4 wherein the target |
nucleotide sequence is a DNA. |
Description |
BACKGROUND OF THE INVENTION |
Surveys of human genomic DNA have indica are present in abundance (Stallings, Gen al., Nucleic Acids Res.,1994. 22(9): p. these sequences has fostered their use i number of human diseases have been shown a subset of these repetitive sequences, 1993, 72(6): p. 971-83; Fu et al., Scien et al., Cell, 1993, 74(1): p. 127-34; Or 221-6; Harley et al., Nature, 1992, 355( 1992, 355(6360): p. 547-8; Aslanidis et La-Spada et al., Nature, 1991. 352(6330) 1991, 338(8762): p. 289-92; Yu et al., S Kremer et al., Science, 1991. 252(5013): 1991, 65(5): p. 905-14; Koide et al., Na |
ted that tandemly reiterated sequences omics, 1994. 21: p. 116-21; Han et 1735-40). The polymorphic nature of n a variety of studies. Recently a to be caused by the expansion of trinucleotide repeats (HDCRG, Cell, ce, 1992. 255(5049): p. 1256-8; Knight r et al., Nat Genet, 1993, 4(3): p. 6360): p. 545-6; Buxton et al., Nature, al., Nature, 1992, 355(6360): p. 548-51; : p. 77-9; Sutherland et al., Lancet, cience, 1991, 252(5010): p. 1179-81; p. 1711-4; Verkerk et al., Cell, t Genet, 1994, 6(1): p. 9-13). |
All of the currently known diseases caus by repeats high in dG+dC (guanine and cy et al., 1994). One method for analyzing amplifying the region using the polymera dG+dC content renders amplification and/ to an increased melting temperature, or of the expanded motif. A common result o repeat motif with a high dG+dC content i tion products which do not correspond to Hum. Molec. Genet., 1993, 2(4): p. 411-1 complicates the interpretation of result have noted the difficulty in interpretin ton's disease (HD) (Riess, O., et al., H Goldberg et al., Hum Mol Genet, 1993. 2( Genet, 1993, 2(6): p. 633-4; Snell et al Barron et al., Hum. Molec. Genet., 1994, |
ed by trinucleotide repeats are caused tosine respectively) content (Han the expansion of such repeats is by se chain reaction (PCR). The high or DNA sequencing very difficult due T.sub.m, and stable secondary structure f amplifying a region containing a s the presence of additional amplifica- the desired product (Hauge et al., 5). Such "stutter" or "shadow" banding s of an assay. A number of authors g the banding patterns seen in Hunting- um Mol Genet, 1993, 2(6): p. 637; 6): p. 635-6; Valdes et al., Hum Mol ., Nat Genet, 1993, 4(4): p. 393-7; 3(1): p. 173-175). |
Several theories addressing the problem been put forth (Litt et al., Biotech., 1 mechanisms resulting in false banding pa annealing to a repetitive sequence or st third explanation proposes that secondar sequences allow the extending DNA strand this were to occur during the early cycl could be made which would eventually app Secondary structure resulting in additio ased stability of a region with an incre stability of base pairs has been a subje Phosphate binding cations have long been of the DNA helix (von Hippel et al., Ann The most likely mechanism for this alter affect that these cations (Cs.sup.+, Li. Mg.sup.++, Ca.sup.++) have on the transf from a non-aqueous to an aqueous environ cations effectively increase the solubil which acts to destabilize the helix in a |
of "stutter" or "shadow" banding have 993, 15(2): p. 280-284). Possible tterns may include improper primer rand slippage during synthesis. A y structure unique to the repetitive to skip cassettes of repeats. If es of a PCR reaction sufficient template ear as additional or "stutter" bands. nal banding may be caused by the incre- ased dG+dC content. The differential ct of inquiry for over three decades. known to be general destabilizers . Rev. Biochem., 1972, 41: p. 231-300) ation of helical stability is the sup.+, Na.sup.+, K.sup.+, Rb.sup.+, er of free energy of a nucleotide ment (von Hippel et al., 1972). These ity of nucleotides in aqueous solutions general fashion. |
Another class of compounds has been show DNA helix based on nucleotide compositio are known to preferentially bind in DNA et al., PNAS, 1973, 70(2): p. 298-302). on the differential levels of hydration the tetraalkylammonium ion being used. P base pairs are more highly hydrated than relatively more suitable binding site fo ions (Tunis et al., Biopolymers, 1968, 6 demonstrated that larger tetraalkylammon of DNA while smaller tetraalkylammonium effect based on base composition (Melchi in this case, is to produce a relative i pairs relative to dG.dC base pairs thus ibution to the T.sub.m of a DNA sequence determining a T.sub.m at which DNA secon use, however, of tetraalkylammonium comp their destabilization effect on DNA-prot tions necessary to achieve DNA isostabil 1993, 32(1): p. 137-44). |
n to alter relative stability of the n. Various tetraalkylammonium ions grooves at dA.dT base pairs (Melchior The mechanism in this case relies between base pairs and the size of revious work has suggested that dA.dT dG.dC base pairs thus providing a r the nonpolar arms of alkylammonium : p. 1218-1223). It has also been ium ions are general destabilizers ions have a differential stabilization or et al., 1973). The overall effect, sostabilization of the dA.dT base eliminating the base composition contr- . Isostabilization is desirable in dary structure would be minimal. The ounds in these studies is offset by ein interactions at the salt concentra- ization (Rees et al., Biochemistry, |
There is a need for a compound which wou of the tetraalkylammonium compounds with effects. |
ld offer the isostabilizing effect out the DNA-protein altering side |
SUMMARY OF THE INVENTION |
This invention relates to an improvement target nucleotide sequence, by using an osmolyte in the reaction mixture of the found that the use of a glycine-based os bands in the amplification product allow nucleotide sequence. For example, detect sequence, indicative of Huntington's Dis of a glycine-based osmolyte. |
of the procedure for amplifying a effective amount of a glycine-based amplification procedure. It has been molyte reduces the appearance of stutter ing for easier detection of the target ion of the target trinucleotide repeat ease, is made clearer with the use |
The present invention further relates to ide sequence for diagnostic analysis, wh osmolyte to be used in the amplification |
a kit for amplifying a target nucleot- erein the kit includes a glycine-based procedure. |
This invention, in addition, relates to nucleotide sequence, the improvement com of a glycine-based osmolyte to the react DETAILED DESCRIPTION OF THE INVENTION |
the improvement of sequencing a target prising adding an effective amount ion mixture of an sequencing procedure. |
This invention is based upon the discove is added to a PCR amplification reaction on's disease the resultant product of th interpretable. The glycine-based osmolyt of the tetraalkylammonium compounds with effects. |
ry that when a glycine-based osmolyte mixture for the detection of Huntingt- e amplification procedure is more e offers the isostabilizing effect out its DNA-protein altering side |
The term "amplifying" refers to the repe nucleic acids (DNA) or ribonucleic acids or non-specific means resulting in an in DNA or RNA sequences intended to be copi rase Chain Reaction (PCR), Nucleic Acid Transcription-based Amplification System ation (3SR), Q-beta replicase, Ligation Chain Reaction (LCR). |
ated copying of sequences of deoxyribo- (RNA) through the use of specific crease in the amount of the specific ed. These processes include the Polyme- Sequence Based Amplification (NASBA), (TAS), Self-sustained Sequence Replic- amplification reaction (LAR) and Ligase |
A glycine-based osmolyte suitable for us trimethylglycine, glycine, sarcosine and |
e in the present invention includes dimethylglycine. |
The term "target nucleotide sequence" re sequence, the presence of which is indic Such "target nucleotide sequences" would otide sequence motifs or patterns specif thereof, nucleotide sequences specific a sequences of interest for research purpo tion to a disease. In general, "target n region of contiguous nucleic acids which technology. |
fers to a portion of a nucleotide ative of a condition, such as a disease. include, but not be limited to, nucle- ic to a particular disease and causative s a marker of a disease, and nucleotide ses which may not have a direct connec- ucleotide sequences" could be any are amenable to an amplification |
The term "sequencing" refers to the copy via biochemical processes. Such "sequenc the deoxyribonucleic or ribonucleic acid sequence and the order in which those nu A typical enzymatic sequencing procedure of contiguous double stranded nucleic ac ent single strands, adding a sequencing the aforementioned region and through th synthesizing a complementary stretch of as Sanger or dideoxy sequencing, a porti of the complementary stretch of nucelic terminate the extension of a nucleic aci run each containing one of the four poss dideoxynucleic acid in a given reaction relative to its comparable deoxynucleic in the sequence. The result is a series depending upon the location at which the By also incorporating a detection system or fluorescent, it is possible to determ in the region in question. |
ing of a target nucleotide sequence ing" refers to the determination of composition of a target nucleotide cleic acids occur in that sequence. would entail the isolation of a region ids, separating them into their compon- primer homologous to a portion of e use of nucleic acid polymerase enzymes nucleic acids. In one scheme known on of the reagents used in the synthesis acids are dideoxynucleic acids which d sequence. Four reactions are normally ible dideoxynucleic acids. As the is present at a low concentration acid it is not used at every occurrence of extension products of various lengths dideoxynucleic acid was incorporated. of some type, typically radioactive ine the sequence of nucleic acids |
EXEMPLIFICATION |
EXAMPLE 1 |
Genomic DNA was isolated from peripheral salt extraction method of Miller et al. extracting DNA from human nucleated cell p. 1215) and resuspended in sterile wate The PCR primer HD17-F3 (5'-GGC GCA CCT G Technologies, Inc. of Alameda, Calif.) ( with fluorescein by the incorporation of synthesis by Operon Technologies. |
blood mononuclear cells by the high (A simple salting out procedure for s. Nucleic Acids Res, 1988. 16(3): r to a concentration of 1 .mu.g/.mu.l. GA AAA GC-3') (purchased from Operon Seq. I.D. No.: 1) was 5' end labeled a fluorescein amidite during HD17-F3 |
Amplification of HD specific sequence wa and HD17-R1 (5'-GCG GCT GAG GAA GCT GA-3 No.: 2) obtained as HPLC purified stocks following: 100-500 ng of genomic DNA, PC KCl, 2 mM MgCl.sub.2) (obtained from Sig (Pharmacia) to a final concentration of was 7-deaza-GTP (Pharmacia), 12.5pM HD17 labelled HD17-F3, 2.5M BETAINE.TM. Mono Sigma Chemical), sterile water. Reaction for three minutes prior to the addition from AmpliTaq, Perkin-Elmer of Foster Ci in a Perkin-Elmer 480 thermal cycler at C., 1 min., 74.degree. C., 1 min. for a products were analyzed using a 6% sequen acia A.L.F. automated sequencer. Sizing to a M13 sequence ladder run on each gel by using the Fragment Manager software p |
s completed using primers HD17-F3 ') (Operon Technologies) (Seq. ID . Each PCR reaction contained the R buffer (IOmM Tris, pH 8.4, 5 OmM ma Chemical of St. Louis, Mo.), dNTP's 200 .mu.M (50% of the dGTP content -R1, 3.1 pM HD17-F3, 9.4 pM fluorescein hydrate (N,N,N, trimethylglycine, tubes were heated to 95.degree. C. of 5 units of Taq polymerase (obtained ty, Calif.). The reactions were cycled 95.degree. C., 1 min., 62.degree. total of 30 cycles. Amplification cing gel containing 8M urea on a Pharm- of bands was accomplished by comparison . Areas under the peaks were determined ackage from Pharmacia. |
Comparisons were made to identical DNA s primers in a PCR reaction mix described between trinucleotide (CAG) repeat lengt disease. Nat Genet, 1993. 4(4): p. 398-4 |
amples amplified with the identical in Andrew, et al. (The relationship h and clinical features of Huntington's 03). |
Table 1 demonstrates the effect BETAINE. The maximum peak was selected for each l each peak using the Fragment Manager sof based on height above a uniform baseline determined by drawing a line from the na region to normalize comparison between c glycine increases the area under the sel samples amplified without N,N,N trimethy when analyzing a normal size HD allele a analyzing HD alleles in the affected ran |
TM. has on amplification of HD alleles. ane by analysis of the area under tware package which selects peaks . The baseline for each curve was dir of one peak to the next nadir urves. The addition of N,N,N trimethyl- ected peak, as compared to identical lglycine, by an average of 9 fold nd by an average of 19.5 fold when ge. |
____________________________________ |
______________________________________ |
Normal Allele Affected Allele |
Fold in |
crease with |
Area under peak Area under peak |
BETAINE |
Sample |
BETAINE |
No Betaine |
BETAINE |
No Betaine |
Normal |
Af |
fected |
____________________________________ |
______________________________________ |
1 732.2 |
45.6 218.2 17.4 16.5 13 |
2 2207 188.4 890.3 33 12 27 |
.5 |
3* 1519.3/ |
413.1/ 4/4.5* |
1212.7 |
286.8 |
4 2654.2 |
322 264.2 15 8.5 18 |
____________________________________ |
______________________________________ |
*Sample taken from an individual wi |
th two normal alleles. Values indicate |
the area under the peak for each no with |
rmal allele and the fold increase |
BETAINE .TM.. |
EQUIVALENTS |
Those skilled in the art will recognize, more than routine experimentation many e of the invention described herein. Such ssed by the following claims: |
or be able to ascertain, using no quivalents to the specific embodiments equivalents are intended to be encompa- |