DE19733619C1 - New PCR (polymerase chain reaction) method for detecting a small number of allelic variations comprises using a combination of PCR and RFLP (restriction fragment length polymorphism) - Google Patents

Abstract

An allele specific method (I), CASE-PCR (combined allele specific enriched polymerase chain reaction), for detecting gene mutations, especially allelic variations which only differ at a few base pairs, is new. An allele specific method (I), CASE-PCR (combined allele specific enriched polymerase chain reaction), for detecting gene mutations, especially allelic variations which only differ at a few base pairs, is new and comprises: (1) performing PCR (polymerase chain reaction) using a complementary wild type analog PNA (peptide nucleic acid) oligonucleotide which suppresses the amplification of surplus wild type alleles along with an oligodeoxynucleotide primer pair; and (2) identifying the mutations or variations using PCR-RFLP (restriction fragment length polymorphism) and a known sequence for a restriction enzyme carrying oligonucleotide.

Description

short version

The invention relates to an enrichment method based on the polymerase chain reaction (PCR) for the detection of allele-specific and point-directed mutations or genetic polymorphisms. It is an improvement of previous methods (PCR-SSCP = polymerase chain reaction with subsequent polymorphism detection via single strand conformational polymorphism), PCR-RFLP = PCR-based detection of a restriction fragment length polymorphism, PCR-ASO = PCR with allele-specific oligonucleotides that The CASE-PCR method is based on two PCR-based techniques, which together allow highly selective enrichment of nucleotide-variant alleles through two interlocking but independent processes:

• 1. Peptide nucleic acid (PNA) mediated selective blockade of PCR ¹ (English PCR clamping) and
• 2. PCR-RFLP to generate a restriction polymorphism and Favoring restriction-resistant heteroduplex DNA fragments in one subsequent restriction digestion.

By combining these methods, a sensitivity of up to 1 variant allele among 10 ³ wild-type alleles is achieved. By limiting the number of completed PCR cycles and by choosing the appropriate PNA and DNA primer in the sense of an offset PCR (English nested PCR), a high degree of diagnostic specificity is ensured at the same time. A further advantage is the possibility of detecting mutations in a section comprising a number of adjacent codons, with RFLP primers being selected depending on the codon.

State of the art Problem

Molecular methods are increasingly used in the diagnosis of malignant tumors used, which have the task of circumscribed specific DNA Detect changes in the area of certain onco- and tumor suppressor genes. The technique on which most of the methods are based offers Polymerase chain reaction (PCR) principally the ability to perform analyzes from the smallest To create tissue samples. However, this is difficult to achieve if the examined ones Gene segments differ only in one or a few bases and also the too investigating alleles in a minority encompassing several powers of ten in biological examination times are available.

Previous methodical verification procedures PNA-mediated PCR "clamping"

This technique is based on synthetic oligonucleotides with DNA-mimicking Characteristics. In addition to ordinary oligonucleotides with ribose phosphate In recent years, synthetic oligonucleotides with an N- (2-aminoethyl) glycine skeleton (so-called peptide nucleic acids, PNAs) related been. These are characterized by a very high binding affinity, which is independent of salt concentrations and even under the high temperatures of the PCR conditions (72 ° C) in the case of 15-mers still forms heteroduplexes. In order to Under these conditions, PNAs are primarily suitable for the protection of DNA Sections by so-called "clamping", i. H. Protection of a relevant gene segment from Replication by DNA polymerases, especially since these are not different from DNA analogues Starter molecules can serve for a PCR reaction (∅rum, H., Nielsen, P.E., Engholm, M., Berg, R.H., Buchardt, O., and Stanley, C. Nucl. Acids Res. 21, 5332- 5336, 1993). Conversely, the avidity is already clear in the case of a point mutation reduced. This difference in the stability of the binding can be exploited in practice by using wild type sequence PNAs together with conventional DNA primers and DNA to be examined the formation of defined Wild-type fragments in the subsequent PCR reaction, but those of mutant alleles allowed. To reinforce the protective effect, an additional one Hybridization step introduced at higher temperature in the PCR program, which a pre-attachment of the PNA molecule, but not the conventional DNA Primer allowed. A variant of the method uses a PNA, which with a DNA primer competes for the target sequence (PCR clamping by primer  exclusion) and successfully used for the detection of circumscribed RAS mutations (Thiede, C., Bayerdörffer, E., Blasczyk, R., Wittig, B., and Neubauer, A. Nucl. Acids Res. 24, 983-984, 1996). Although the process is relatively easy to do, however, there is no possibility of receiving fragments as safely mutated fragments to be distinguished from normal alleles, which are due to incomplete blockade by PNAs arise, apart from the possibility of sequencing them individually. In addition is successful detection of the stoichiometry of the initial parameters (quantity used DNA, DNA primer etc.) dependent and therefore prone to failure, so here often false positive fragments are obtained.

PCR-RFLP

This PCR method has been published by various authors as a possibility of Detection of circumscribed allele variants using restriction length polymorphism (Kahn, S.M., Jiang, W., Culbertson, T.A., Weistein, I.B., Williams, G.M., Tomita, N., and Ronai, Z. Oncogene 6, 1079-1083, 1991; Levi, S., Urbano-Ispizua, A., Gill, R., Thomas, D.M., Gilbertson, J., Foster, C., and Marshall, C.J. Cancer Res. 51, 3497- 3502, 1991; Jacobson, D.R. and Mills, N.E. Oncogene 9, 553-563, 1994; Mills, N.E., Fishman-C. L., Scholes, J., Anderson, S.E., Rome, W.N., and Jacobson, D.R. J. Natl. Cancer Inst. 87, 1056-1060, 1995). As a rule, to simplify detection necessary palindromic sequence for a restriction endonuclease in the Examining section does not exist, such is by point-directed Change one of the two primers (so-called "mismatch" primer) into the resulting PCR Product introduced. The interface to be introduced is chosen so that it The codon to be examined includes and natural mutations in this section cause a change in the motif. The subsequent restriction end nuclease digestion cleaves wild-type fragments, but not mutated alleles. By repeated PCR amplification followed by restriction endonuclease digestion highly selective enrichment of mutant alleles can also be achieved. This However, the procedure is relatively time-consuming and associated with the risk of having to Generate non-specific mutations as part of the additional amplifications.

PCR ASO

Allele-specific PCR using allele-specific oligonucleotides. The selection mutated alleles are carried out by mutation-analogous complementary primers. These will as in a conventional PCR reaction in one or two successive PCR Steps used, the hybridization necessary for primer attachment temperature is chosen so that heteroduplex formation is possible only with those Mutant-bearing variant alleles, but not with normal alleles. disadvantage this method is essentially the slight difference between the  Optimal temperature of the wild type and the mutant allele, which often makes it wrong positive amplificates are created.

Description of the procedure

The invention relates to a combination method, the CASE-PCR = "combined alleles specific enrichment-PCR ", which is a simplified PCR method for selective enrichment and for the specific detection of variant alleles (shown schematically in Figure 1).

The claims do not refer to the actual PCR process itself, so far as it is protected by U.S. Patent 4,683,195.

Phase sequence

The method consists of two successive PCR methods, in the first step (enrichment step) of a PCR clamping reaction using the following components:

• - An opposing PNA oligonucleotide covering the mutation area 15 bp in length,
• - a conventional oligodeoxynucleotide primer pair (two the 5 'and the 3 'region of a DNA segment delimiting oligonucleotides = ODNs) for Amplification of a gene fragment of interest,
• - The DNA sample to be examined, which in addition to the normal variant (= Wild-type allele) shares one in one or a few base pairs distinguishing point mutated variant contains
• - Taq polymerase,
• - PCR buffer with all necessary reaction components

The actual PCR reaction sequence comprises four segments per cycle:

• 1. Denaturation at 94 ° C
• 2. PNA hybridization at approx. 70-78 ° C
• 3. Primer hybridization at approx. 56-64 ° C
• 4th elongation step at 72 ° C

After 25-35 cycles have been completed, a sample of the PCR mixture (2 µl of 25 or 50 µl) is used for the subsequent PCR-RFLP reaction (diagnostic step) This consists of:

• - a false base primer, which is complementary to the mutation-carrying section is and a restriction polymorphism in the area of the to be examined Generated codons
• - an associated 3 'primer (can be identical to the 3' primer of the first PCR Reaction)
• The DNA sample to be examined from the first PCR clamping reaction, Taq Polymerase,
• - PCR buffer with all necessary reaction components

The actual PCR reaction sequence comprises three segments per cycle:

• 1. Denaturation at 94 ° C
• 2. Primer hybridization at approx. 56-64 ° C
• 3rd elongation step at 72 ° C

After going through about 25 amplification cycles, a sample amount (10 µl) is digested with the restriction enzyme associated with the palindrome generated. The reaction approach for this contains:

• Reaction buffer with a salt concentration specific for the enzyme,
• - sample amount of the DNA fragment obtained (approx. 1 µg),
• - 10-30 units of the respective restriction endonuclease

Incubation takes place at 37 ° C for 1-3 h in a corresponding heating block. After successful digestion, the PCR fragments obtained are gel electrophoresis separated in a 1% agarose gel by means of ethidium bromide staining under UV Suggestion made visible, then photo-documented.

Advantages and innovations of the process

These result from the strongly selective allele enrichment via PCR clamping combined with the only slightly selective (by preference so-called heteroduplex fragments) but diagnostically clear PCR-RFLP. By combining both steps, the DNA amplification required for detection can be achieved in one process with the necessary allele enrichment in only approx. 50-60 PCR cycles; the choice of two conventional primer pairs also provides the necessary security of a PCR with offset primers by preventing the formation of non-specific fragments by independently hybridizing oligonucleotides in each step. The quality characteristics can be summarized as follows:

• - highly selective allele-specific enrichment,
• - Clear diagnostic statement about the generation of a codon-related Restriction polymorphism with separation of the mutated allele fraction from residual wild-type fragment in the subsequent gel analysis,
• - Limitation to a relatively small number of PCR cycles, thereby extensive avoidance of artificial in vitro mutations by the Taq Polymerase itself,
• - exclusive amplification of specific fragments by using "Nested" primers, thereby additional diagnostic security
• - largely independent of the exact amount of DNA in the examination materials as well as defined PNA and DNA primer ratios.

5. Examples of use 5.1 DNA-based CASE-PCR

This is based on circumscribed mutations in the region of a gene, which usually comprise only one nucleotide. These include "hot spot" mutations of a number of onco or tumor suppressor genes. Examples of this are (taken from C. Wagener, Introduction to Molecular Oncology, Thieme Verlag Stuttgart, New York, 1996):

RAS: Codon 12, 13, 61 (for the N, H, and K-ras genes)
p53: codons 175, 245, 248, 249, 273, 282
FAP: Codon 1309, 1378, 1450, 1464, 1487-90
ARP: Codon 50
RET: Codon 908 (Mulligan et al., Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A, Nature 363 (1993), 458-461).

DNA-based CASE-PCR offers all the advantages of DNA-based PCR diagnostics of cytomaterial:

• - Independence from the cytological quality
• - Determination from small amounts of sample
• - no further sample preparation, cold chain or storage problems

5.2 RNA-based CASE-PCR

This includes the detection of defined deletions in an RNA section or the Expression of an alternative exon in a percentage different composite RNA population. For this, the cell material is first under Cooled centrifuged and the pellet subjected to an RNA extraction procedure. The conversion to reverse cDNA is also carried out according to standard protocols. On PNA homologous to the deletion area or the area of the additional exon Oligonucleotide largely suppresses one controlled by external DNA primers Amplification of a section of cDNA. By using a second pair of DNA primers and exploitation of a natural or newly generated polymorphism in this In the area, wild-type fragments can easily be distinguished from variants.

5.3 CASE-PCR in the diagnosis of malignant diseases

The need for efficient tumor diagnostics arises in connection with the clinical question regarding the presence of malignant cells in fluid, cytology or tissue samples. Since the proportion of mutated oncogenes and suppressor genes in solid tumors of all entities is usually relatively high, a correspondingly sensitive PCR method, which shows the detection of specific lesions, could be applied to a large area of tumor diagnosis. This concerns above all the early detection in the case of

• - gastrointestinal tumors (colorectal tumors, pancreatic carcinomas, Biliary tract, bladder cancer)
• - tumors of the oropharynx area, v. a. of bronchial carcinoma
• - Carcinomas of the urinary tract
• - Uterine and cervical cancer

These tumors are characterized by the fact that they are usually connected to natural ones Have derivation paths, so that a determination from exfoliative material without invasive Intervention is possible. In addition, the determination of genomic mutations is also over Cell material from diagnostic punctures, in particular from pleural and Ascite punctures, also possible from bone marrow biopsies.

More applications

Contamination problems with homologous DNA from cell material (e.g. cell separates etc.)  Allele and individual-specific detection by enrichment from DNA and / or Mixed cell samples

Further development of the process

The following further developments, especially with regard to commercialization, are possible within the scope of the CASE-PCR technique:

• - Further development in the sense of "multiplex" PCR through simultaneous use different PNA and DNA oligonucleotides for analysis of multiple Mutations
• - Standardization of reagents, oligonucleotides, reaction sequence and -temperature to simplify the process
• - Development of a solid phase test, for example in analogy to PCR-ELISA procedures

Illustrations

Fig. 1: Representation of the CASE-PCR method with the two enrichment steps and verification by restriction endonuclease digestion

Fig. 2: Temperature profile during PCR clamping and the PCR-RFLP reaction

Claims (2)

1. Allele-specific enrichment methods for the detection of gene mutations or allelic variants which differ in only a few base pairs, characterized in that

• a) a complementary wild-type analog PNA oligonucleotide for suppressing the amplification of excess wild-type alleles is used together with an oligodeoxynucleotide-primer pair (ODNs) in the PCR reaction and
• b) the mutations or allelic variants are identified with the aid of a PCR-RFLP step with allelic variants and a recognition sequence for oligodeoxy nucleotides carrying restriction enzymes.

2. The method according to claim 1, characterized in that solid phase coupled Primers and / or PNA molecules can be used or multiple combinations of primers in the sense of a multiplex PCR.