FR2722295A1 - Single strand conformation polymorphism analysis of DNA - Google Patents

Abstract

In a single strand conformation polymorphism (SSCP) analysis of DNA, the new feature is the use of an electrophoretic gel of low ionic strength. Also new are:(1) disposable, preformed electrophoretic gel for this process prepd. with a buffer of low ionic strength, and(2) kits for the process comprising such a gel and a migration buffer of low ionic strength.

Description

The present invention relates to a method for analyzing polymorphisms of

  single-stranded conformation of DNA also called SSCP (Single-Strand conformation polymorphism analysis) method Small variations in the nucleotide sequence of a gene make it possible to distinguish between abnormal cells and normal cells in patients suffering from a disease linked to hereditary or sporadic changes in the genome. Thus it is known that the accumulation of several genetic modifications at the level of oncogenes or tumor suppressor genes are necessary for the genesis of cancers in humans. These changes in DNA seen in cancer cells can be alterations affecting large areas of the genome, such as amplifications, rearrangements and loss of genes, or changes in DNA can affect short nucleotide sequences. These are in particular single base substitutions, deletions or insertions of one or a few nucleotides. Alterations affecting large regions can be detected by simple hybridization in uSouthern blot ". However, few simple, reliable and rapid methods exist for the detection of

occasional transfers.

  The SSCP analysis method described by Orita et al. (1,2) allows

  highlight point mutations.

  In the SSCP method two complementary single-stranded sequences

  are separated by non-polyacrylamide gel electrophoresis

  denaturing. Differential migration is a function of the tertiary structure, and therefore of the nucleotide sequence of each strand. Single-stranded DNA fragments of the same length, differentiated by the substitution of a base, have different electrophoretic migrations in non-denaturing polyacrylamide gel. The difference in mobility is attributed to a difference in tertiary conformation, which is changed by changing a single base. We call this

  characteristic: single-stranded conformation polymorphism or SSCP.

  The combined use of PCR and SSCP analysis therefore constitutes

  a simple and sensitive method for the detection of point mutations.

  This method is called PCR-SSCP.

  In this PCR-SSCP method, the amplified material is heated to denature the double-stranded DNA and then loaded onto a polymer gel such as non-denaturing polyacrylamide. The mobility modifications of one or both complementary strands visible on the autoradiography, reveal the presence of a basic substitution on one of the alleles. PCR-SSCP analysis is a simple and sensitive method for detecting changes in the nucleotide sequences of genomic DNA and cDNA. It is possible to detect basic substitutions, insertions

  or short sequence deletions as well as allelic gene loss.

  This method is therefore suitable for the analysis of DNA and RNA for

  cancers and genetic diseases in humans.

  One of the advantages of the PCR-SSCP technique is in fact the sensitivity reached by the test since DNA alterations can be detected from a very small number of affected cells in the sample. A substitution of a base on one of the alleles is visualized by a modification of the strand mobility. PCR-SSCP analysis can also detect the loss of genes. Indeed, if modifications of mobility linked to a polymorphic point mutation can be observed on the DNA of normal cells, a disappearance of these signals (generated by one of the two alleles) on tumor cells of the same individual indicates a loss of an allele of the gene in the tumor. The absence of signals corresponding to a normal sequence therefore indicates the loss of one of the alleles. A PCR-SSCP analysis can therefore reveal two types of genetic modification in tumor cells: loss of one allele and mutation on the remaining allele. PCR-SSCP analysis is also capable of detecting mutated mRNA. To do this, the mRNA extracted from cells is converted into cDNA using

  a reverse transcriptase, then this cDNA is analyzed by PCR-SSCP.

  PCR-SSCP analysis has been used to detect DNA damage in human cancers; mutations activating the ras gene in lung cancers and mutations inactivating tumor suppressor genes, such as RB and P53, in different varieties of cancers. The SSCP method has also played an important role in identifying the genes responsible for hereditary diseases in humans: the CFTR gene (for "Cystic Fibrosis Transmembrane conductance Regulator ') responsible for cystic fibrosis, the neurofibromatosis type gene

  1 (NF1) and the gene for colonic adeno-polyposis (APC).

  The PCR-SSCP method is therefore particularly effective in detecting any variation of the order of a few bases in the DNA sequence, among which the substitutions of a base which are difficult

  detected by other techniques.

  Another definite advantage of the PCR-SSCP analysis is that one obtains, at the end of the experiment, a physical separation of the mutated fragments which can then be purified. After autoradiography of the dry gel, a tiny fragment of the gel, corresponding to the band of interest, is cut out, the DNA is extracted therefrom, then amplified by PCR. The sequencing analysis of this PCR product ultimately identifies the mutation

on the target gene.

  The sensitivity of the PCR-SSCP method in principle allows the detection of point mutations in a DNA fragment up to 900 base pairs. However in practice the effectiveness of the method for the detection of point mutations for fragments of more than 300

  base pairs is less than 90% (4).

  Thus, it has been described that 100% of the mutants of the mouse beta-globin gene have been detected on DNA fragments of 193 bp (8), while 87 96 of the mutations were detected only in fragments of 354 bp (4). Efficiency of 90% has been reported on p53 gene fragments from 202 to 309 bp. To date, it is therefore recommended to perform a PCR-SSCP analysis on fragments below 300 bp to guarantee a

100% reliability.

  Electrophoretic media and electrophoresis gels prepared with acrylamide type monomers have been described in the application for

European patent EP 0339 678.

  It has been discovered according to the present invention, that the ionic strength of the composition of the electrophoresis gel used for carrying out the SSCP analysis affects the mobility of single-stranded DNA. More particularly, it has been discovered that the reduction in the ionic strength of the composition of the gel considerably improves the resolution of SSCP, that is to say the capacity for electrophoretic separation of two strands of DNA of the same size which are distinguished. by a simple substitution

of a base.

  The present invention therefore demonstrates that the opinion accepted to date according to which the electrophoretic separation observed is linked to the only difference in tertiary conformation adopted by DNA strands.

folded is not exact.

  The present invention thus provides improvements to the SSCP analysis method which make it possible to detect 100% of minor genetic alterations, including point mutations, deletions or short insertions, in fragments, more than 300 in particular, from 300 to

500 nucleotides.

  More precisely, the subject of the present invention is a method for analyzing polymorphisms of single-stranded conformation of DNA, called SSCP, characterized in that a low electrophoresis gel is used.

ionic strength.

  In particular, the electrophoresis gel is prepared using a

low ionic strength buffer.

  Preferably, a low migration buffer is also used.

  ionic strength, when performing electrophoresis.

  The expression “weak ionic strength” is understood here to mean a molar concentration of gels reduced compared to the concentrations described in the literature for performing SSCP electrophoresis with the gel and / or the buffers of

migration in question.

  In one embodiment the electrophoresis gel is prepared

  with TBE buffer (Tris-Borate, EDTA).

  Similarly, in one embodiment the migration buffer is a

TBE buffer (Tris-Borate, EDTA).

  The TBE buffer is the most commonly used electrophoresis buffer. Indeed, this pad is particularly resistant to the Joule effect

  intervening during migrations under high tension.

  In both cases, TAE (Tris-Acetate, EDTA) can also be used.

  When the intrinsic buffer entering into the composition of the gel is TBE, a buffer diluted more than 1/2 (less than 0.5 X) to 1 / 10th (0.1 X) relative to the concentration of TBE is used. 1 X (0.09 M Tris-base, 0.09 M Boric Acid and 0.002 M EDTA). We use in particular a

TBE buffer (0.4 X).

  In the intrinsic TBE buffer, therefore, the expression “low ionic strength” is understood to mean a molar concentration of negative borate counterions.

less than 45 mM.

  Likewise, suitably, the migration buffer is the same TBE 1 X buffer (Tris-Borate 0.09 M and EDTA 0.002 M) with molar concentration diluted 1 / 10th (0.1 X) to 1 / 20th ( 0.05 X). In the TBE migration buffer, therefore, the expression “low ionic strength” is understood to mean a molar concentration of borate ions of less than 9 mM. Conventionally, the electrophoresis gel is a polyacrylamide gel prepared with acrylamide type monomers,

  especially acrylamide and bisacrylamide.

  Preferably when the gel is a polyacrylamide gel, it is a gel with a final polyacrylamide concentration of 2 to 12%, in particular 6 9 The present invention also relates to a preformed electrophoresis gel for single use useful for implementing a method according to the invention, characterized in that it is prepared for

  from a buffer of low ionic strength, as characterized above.

  Finally, the subject of the present invention is a kit of reagents for the implementation of an SSCP electrophoresis method according to the invention comprising a liquid gel ready to be polymerized according to

  the invention and a migration buffer as characterized above.

  Other characteristics and advantages of the present invention will appear in the light of a detailed embodiment which shows an efficiency of 100 96 in the detection of mutations in fragments from 204 to 358 bp as evidenced by comparison between the data of

direct sequencing and SSCP.

  FIG. 1 shows that the modification of the composition of the gel and of the migration buffer according to the present invention considerably increases the sensitivity and the amplitude of the movements of the bands.

from SSCP.

  (A) = conventional gel and (TBE 1X) and migration buffer (TBE 1X) and; (B) = gel (TBE 0.4X) and migration buffer (TBE O, lX) force

reduced ionic.

  Figure 2 shows the detection of a single substitution

  nucleotide in a 341 bp fragment of the APC gene.

  1. Material and method The DNA used was obtained from tumor fragments or from PBMC lymphocytes from patients by the proteinase K digestion method and phenol / chloroform extraction. The primers used for the PCR amplification were designed using the software OLIGO Société MEDPROBE, Oslo, Norway -. They make it possible to amplify all the exons of the p53 gene and thus to generate fragments from 204 bp (exon 7) to 358 bp (exon 4). The entire coding region of the APC gene has been divided into 41 segments between 198 and 356 bp. For these two genes, each segment was amplified separately using the

following PCR protocol.

  ng of genomic DNA are amplified with dNTPs 5 & M (Pharmacia), Mgc12 1.25 to 1.5 mM (Perkin), PCR buffers 1X (Perkin), 3 picomol of each primer, 2liCi of a33P dATP (Amersham ) and

  0.5 units of Taq polymerase (Perkin) in a reaction volume of 20 gll.

  The amplification is carried out in 30 cycles of 30 s at 94 C, 30 s at 56 C (for the p53 gene) or 52 C (for the APC gene) (APC = adenomatous polyposis coli) and sà72 C. After the last cycle , 1 to 2 kl of amplification product are diluted

  in 9 11 of buffer solution A (0.1% SDS and 10 mM EDTA / pH 8).

  Then, 10.1 l of buffer solution B (95% deionized formamide, 0.05% bromophenol blue, 0.005% xylene cyanol and 20 mM EDTA / pH8) are added to the diluted PCR products. The mixture is then denatured by heating at 90 ° C. for 2 minutes and then placed in ice for 3 minutes before being deposited on two gels: - one with glycerol for migrations at room temperature;

  - the other without glycerol for migration at 4 C.

  A hydrolinkTM MDETM gel and an equivalent 6% polyacrylamide gel were used at a polymer concentration equivalent to that proposed by the manufacturer (BIOPROBE). However, the ionic strength of the composition is reduced because the TBE buffer used is at an

  concentration of approximately 0.40X (instead of 0.6X) for the preparation of the gel.

  More specifically, TBE 0.38X is used for the gel without glycerol intended for migration at 4 ° C. and TBE 0.41X is used for the gel with glycerol intended for migration.

migration at room temperature.

  The other components for the preparation of the gel are used in

  the conditions recommended by the manufacturer (see Table 1).

  The composition of the TBE 1OX buffer is as follows: - Tris 108 g (0.9 M); Boric acid 55 g (0.9 M); -EDTA - 0.5 M - pH8 40ml (0.02M); -H20 (qs) il TBE 1X = 1 / 10th dilution of TBE 10X, i.e. the following final concentrations - Tris 90 mM - Boric acid 90 mM -EDTA, pH8 2mM Another modification related to the reduction of the ionic strength of electrophoresis buffer or migration buffer which is 0.05 X to 0.1 X

  TBE instead of a TBE of 0.5 X to 1 X of TBE used in literature.

Table 1

COMPONENTS GEL 4 C GEL 20 C

  without glycerol with glycerol Hydrolink MDETM gel or acrylamide gel * 17.5 ml 17.5 ml TBE 10 X 3 ml (0.38X) 3.5 ml (0.42X) Glycerol 0 6.4 mi H2O ultrapure 57 ml 57 ml 10% ammonium persulfate solution 300. ' 3001M Temed ** 45g TOTAL 78 ml 85 mi *: stock solution of 30% acrylamide with an acrylamide / bisacrylamide ratio of 29/1 ** Polymerization agent The use of a 6% polyacrylamide gel polymerized with persulfate ammonium and TEMED (tetraethymamine diamine) instead of the hydrolink MDETM gel under the same conditions, gave exactly the

same results.

  Preparation of the gel In a beaker containing deionized water, the polymer solution and the TBE buffer are mixed. Then add TEMED, if necessary glycerol, and the ammonium persulfate solution. Then we mix. The mixture is introduced at the end of the electrophoresis plates so as to make the mixture flow between the plates. We wait about 1 hour

  polymerization before starting electrophoresis.

  Electrophoresis device: Electrophoresis is carried out with a device consisting of two glass support plates which are degreased and then assembled. Place a comb at one end of the support and pour the gel into the space

  formed between the two glass plates at room temperature.

  The electrophoresis of the gels was carried out at 10 Watts for 16 hours. The gels are transferred to Whatman paper, left to dry for 30 minutes and then exposed to autoradiography for 2 to 12 hours.

without intensifying screen.

  2. Results 2.1. SSCP analysis of p 53 samples A large number of DNA samples were tested in parallel

  as for their mutations in the p53 gene by sequencing and by SSCP.

  All the negative results by SSCP correspond to wild type sequences, determined by direct sequencing carried out on PCR products (single-stranded DNA). All samples displaced by SSCP correspond to single base substitutions, which are either true mutations or polymorphisms. The correlation between SSCP and direct sequencing is 100% for both

  negative samples (N = 539) and positive samples (N = 70).

  Identical mutations or polymorphisms have been detected in

  several samples showing the reproducibility of the method.

  Positive results corresponding to variations in the

  p53 gene sequence (N = 30) are presented in Table 2 below.

  These results indicate that the SSCP method according to the invention

  can detect any modification of the p53 gene sequence.

  Table 2: detection by SSCP of variations in the sequence of the p53 gene identified by sequencing: T = DNA extract from tumor Ly = Extract of lymphocytes nt = nucleotide + to +++ = intensity of displacement bp = base pair 2.2. SSCP analysis of the APC gene Analysis of the APC gene makes it possible to detect the genetic predispositions for FAP (Familial Adenomatous Polyposis). Given the length of the gene which makes systematic sequencing very difficult, the SSCP method is the only detection method

  possible, the results of which are presented in Table 3.

  Here again, a 100% correlation is obtained between the SSCP method according to the invention and direct sequencing. For each band we associate a genetic event such as a point mutation, a deletion or insertion. The sensitivity of the method makes it possible to detect the substitution of a single nucleotide on a fragment of 341 bp. The sequence of the displaced sample was altered (CAG -> TAG at nucleotide 2101) with the appearance of a nonsense codon at the

  position 696 of the APC gene (Figure 2).

   A 'Cs CDi i ++++ ML <- YI) stZ dq t'OZ LL SbZ IS ++++ DV <- MvDJ SbZ dq bOZ L 1 I [ILS ++++) W <- DVI 17úZ dq tOZ L X'l II] .LIII ++ oL <-ivi 0OZ dq úú 9 L LL iS + ± DLL <- 9LD 91Z dq úú 9. L6ZIHS +++ 99D <-VDD úIZ dq lúú 9 IL.D ++ VJD <- VOD% I dq 1úú 9.L 91 3S +++ mDV <- DXL 9LI dq ú0 S 1, 06S LHS +++ VDD <-!) DD úS dq ú0ú 'S'1 Z} t [iS ++ + uolijlap dq I ISI dq ú0ú S '' II IfID + DLD <-D99 SOi dq gSú b I LZ EIS +++ M0 <-DM3 ZL dqs fS b L Sú EIS ++ D <-O LZ71I1U dq IZ Z uo. lul X1L6Z NV dDSS a: uanbs el úSd auld; uaulleJd úsd aud uoOlllueq) g, .l ap uoljal,) a1 sup uopp uoli uopo np auoo p lllel np uoxg uolJel! Jluapl Z nealqe.l

c> J C> J - -

  ++ & <-VO / LL / 1 U dq 861 0 uoalul q b9S NGIV +++ Vu <- VDD Zbú dq 61 01 -l ZI NGV ++ mI .. <- LL U8úú dq 861 01 [<SIL NUV - D3 <-J, 99L'l lu dq 8úZ 6 uoJlul 'I 90oú NOV +++, 99L <-9XD Z8Z dq SSZ' ú 8Z LS +++ DV "D <- DV [gZdq: SSZ 8 0SúZ +++ VD <- VD9V 0Z dq SSZ L ZI ES +++ 1, L <-IDD 8LZ dq SSZ 8 1 68S IS ++ JD9 <- J3D PLZ dq SSZ 8 J, Ltl rIS +++. 0V <-LDD úLZ dq SSZ 8 S lllD +++. IUtL <-I, 99 fLZ dq SSZ 8 t HIl) +++, l, O <-, IDD úLZ dq SSZ 8 'lg 8S NC1V +++ alLV <- OLD ZLZ dq SSZ 8 ú UnD ++++ VVV <- WVVD SZ dq boZ L 8SZ AS ++++ IOV <- 99V 6bZ dq 'OZ L 1 6Z +++ 1OEL <- DD 8bZ dq bOZ L 1 ZIS uIS ++++ DVD <- 99D 8tZ dq tOZ L 1 L HS dDSS aauanbps el úSd auge luamlsl ú d au? Uoll! Lueq9, jlap uolJ: alD? Suep uolleJlEA np uopoD np aIIe.L np uox 'uo! JI43ujluapl w on

Table 3

  Ln Identification Exon of the Gene Size of the Variation of eN of the Sample APC Fragment Sequence SG 39 3,298 bp AGA -> TGA at nt 376: Arg-> Stop SG 91 3,298 bp TAT-> TAG at nt 306: Tyr -> Stop SG 39 5 349 bp A -> T in intron 4 SG 117 9.1 299 bp 2 bp deletion at nt 1116 SG 9 9.2 237 bp GAG -> TAG at nt 1282: Glu -> Stop SG 57 11 325 bp C -> T at nt 1476: silent SG IOIT 11 325 bp I bp deletion in intron 11 SG 115 12 198 bp G -> A at nt 1572: silent SG 176 13 254 bp CGA -> TGA at nt 1707: Arg-> Stop SG 135T 13 254 bp CGA -> TGA at nt 1678: Arg -> Stop IGR 252 14 354 bp 4 bp deletion at nt 1892 SG 61 15-1 341 bp CAG -> TAG at nt 2101: Gln -> Stop SGC 1OlT 15-1 341 bp A-> T in intron 14 SG 21 15-2 316 bp CGA -> TGA at nt 2431: Arg-> Stop SG 111 15-3 262 bp 4 bp deletion at nt 2562 SG 255 15-3 262 bp GAA -> TAA at nt 2695: Glu-> Stop SG IO1T 15-3 262 bp CGA-> TGA at nt 2644: Arg-> Stop | no ur RN Table 3 (continued) Ln on N Identification Exon of the Gene Size of the Variation of cqN of the echan tillon APC Fragment Séquence SG 329 15-5 276 bp CCA -> CCG at nt 3021: silent SG 32 15-5 276 bp 1 bp deletion at nt 3095 LOVO 15-6 260 bp CGA -> TGA at nt 3358: Arg -> Stop SG 34 15-7 330 bp 4bp deletion at nt 3462 -t SG 188 15-9 312 bp 5 bp deletion at nt 3945 SG 262 15-9 312 bp CAG -> TAG at nt 4030: Gin -> Stop SG 314 15 -9 312 bp 5 bp deletion at nt 3938 SG 101 T 15-9 312 bp CGA -> GCG at nt 3993: silent LOVO 15-10 331 bp 1 bp deletion at nt 4308 SC, 135T 15- 10 331 bp AGT-> AT at nt 4262: Ser-> lie SG 135T 15-11 268 bp 2 bp insertion at nt 4412 SG 205 15-11i 268 bp ACG -> ACA at nt 4497: silent CO10205 15-12 324 bp 1 bp insertion at nt 4685 SG 32 15-13 305 bp CAT-> GAT at nt 4903: Hlis-> Asp SG 129 15-15 316 bp GAC-> GTCat nt 5483: Asp-> Val SG 22 15-17 356 bp 1 bp deletion at nt 6068 Lio CD tii 1-4 i A rn

BIBLIOGRAPHY

  1. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., & Sekiya, T. Detection of polymorphisms of human DNA by gel electrophoresis as singlestrand conformation polymorphisms. Proc. Natl. Acad. Sci. USA 86,

2766-2770 (1989).

  2. Orita, M., Suzuki, Y., Sekiya, T. & Hayashi, K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the

  polymerase chain reaction. Genomics. 5, 874) 879 (1989).

  3. Cai, Q-Q & Touitou, I Excess PCR primers may dramatically

  affect SSCP efficiency. Nucleic Acids Res. 21, 3909-3910 (1993).

  4. Fan, E., Levin, D.B., Glickman, B.W. & Logan, D.M. limitations

  in the use of SSCP analysis. Mutat. Res. 288, 85-92 (1993).

  5. Michaud, J., Brody, LC., Steel, G., et al. Strand-separating conformational polymorphism analysis: efficacy of detection of point mutations in the human ornithine-d-aminotransferase gene. Genomics 13,

389-394 (1992).

  6. Condie, A., Eeles, E., Borresen, AL, Coles, C., Cooper, C., & Prosser, J. Detection of point mutations in the p53 gene: comparison of single-strand conformation polymorphism, constant denaturant gel

  electrophoresis, and hydroxylamine and osmium tetroxide techniques.

Hmm. Mutat. 2, 58-66 (1993).

  7. Glavac, D. & Dean, M. Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point

  mutation. Hmm. Murat. 2, 404-414 (1993).

Claims (12)

  1. Method for analyzing polymorphisms of single-stranded DNA conformation, known as the SSCP method, characterized in that a   low ionic strength electrophoresis gel.   2. Method according to claim 1, characterized in that   uses a low ionic strength migration buffer.   3. Method according to claim 1 or 2, characterized in that the non-denaturing electrophoresis gel contains an intrinsic buffer of weak ionic strength.   4. Method according to one of claims 1 to 3, characterized in   that the electrophoresis gel contains a TBE buffer (Tris-Borate, EDTA).   5. Method according to one of claims 2 to 4, characterized in   what the migration buffer, is a TBE buffer (Tris-Borate, EDTA).   6. Method according to one of claims 1 to 5, characterized in   what the electrophoresis gel, is a gel prepared with an acrylamide polymer. 7. Method according to claim 6, characterized in that the   polyacrylamide concentration is 2 to 12%.   8. Method according to claim 4, characterized in that the   TBE buffer used in the gel composition is a 1 X TBE buffer (Tris-   BORATE 0.09 M and EDTA 0.002 MNI) diluted more than 1/2 (less than 0.5 X) at 1 / 10th (0.1 X).   9. Method according to one of claims 2 to 8, characterized in   what the migration buffer is 1 X TBE buffer (0.09 M Tris-Borate and   EDTA 0.002 M) diluted 1 / 10th (0.1 X) to 1 / 20th (0.05 X).   10. Preformed disposable electrophoresis gel useful for   implementation of a method according to one of claims 1 to 9,   characterized in that it is prepared from a pad with low ionic strength. 11. Electrophoresis gel according to claim 10, characterized in that it comprises: - a polyacrylamide polymer at a concentration of 2 to 12% and;   - a TBE buffer according to claim 8.   12. Reagent kit for implementing a method according to   one of claims 1 to 9, characterized in that it comprises a gel   according to claim 10 or 11 and a migration buffer according to claims 2, 5 or 9.