Many single-base substitutions of base pair leading to inherited diseases, the predisposition to genetic disorders, and cancer are increasingly being discovered. The ability to amplify specific DNA sequences by the polymerase chain reaction (PCR) has made it possible to rapidly and accurately diagnose many inheritable diseases.
Prior to the use of PCR, point mutations were identified by using direct cloning and sequencing, Southern blotting and hybridization with labeled oligonucleotide probes centered on the site of the mutation or digestion with restriction endonucleases. These methods have been greatly enhanced by PCR, which allows amplification of DNA fragments containing the polymorphic sites from minute quantities of DNA. However, these techniques tend to be time-consuming, complex, require the use of a radioactive label, and, in the case of the restriction endonuclease detection, are only applicable when the mutation alters a known cleavage site.
Polymerase chain reaction using allele-specific oligonucleotides (ASOs) is an alternative method for the detection of mutations in which only the perfectly matched oligonucleotide is able to act as a primer for amplification. The advantage of ASO PCR is that it is a rapid, simple, and nonradioactive method. ASO-PCR, otherwise known as the amplification refractory mutation system (ARMS) was first described for the detection of mutations in the α1-antitrypsin gene. It has since been adopted in the study of a number of genes, including prenatal diagnosis of cystic fibrosis, polymorphisms of apolipoprotein E, and point mutations in the ras oncogene.
In this technique oligonucleotide primers are designed such that they are complementary to either the normal (wild-type) or mutant sequence, and both are used in conjunction with a common primer. Because DNA polymerase lacks a 3′ exonuclease activity, it is unable to repair a single-base mismatch between the primer and the template at the 3′ end of the DNA primers. Thus, if oligonucleotide primers are designed to contain mismatches close to or at the 3′ end, the primer will or will not be extended depending on which alternative single-base polymorphisms are present in the target sequence. Hence, under the appropriately stringent conditions, only target DNA exactly complementary to the primer will be amplified.
Below is a simple protocol for allele-specific nucleotide PCR.
All reagents should be of molecular biology grade and solutions made up with sterile distilled water.
- PCR reaction buffer (10X): 100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM
MgCl2, 0.01% gelatin. Autoclave and store at –20°C (see Note 1).
- Nucleotide mix: 200 mM each of dATP, dCTP, dGTP, and dTTP in sterile distilled water and store at –20°C.
- Allele-specific oligonucleotide primers and common primer: 10 μM. Store at –20°C (see Note 2).
- DNA Taq polymerase.
- Sterile distilled water.
- Sample DNA (see Note 3).
- Mineral oil.
- To 0.5 mL Eppendorf tube, add 5 μL 10X reaction buffer, 5 μL nucleotide mix, 5 μL ASO primer (either wild or mutant type primer) (see Note 4) and 5 μL common primer, 100 ng template DNA, and 2 U Taq polymerase (see Note 5), makeup to a final volume of 50 μL with sterile distilled water (see Note 6) and overlay with mineral oil to prevent evaporation.
- Place Eppendorf tubes on the thermal cycler to amplify the DNA by repeated cycles of denaturation, annealing, and extension: initial denaturation of 94°C for5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min (see Note 7).
- Following thermal cycling, electrophoresis 10 μL of the reaction sample through an agarose gel, with a DNA size marker and stain with ethidium bromide. A typical result is shown in Fig. 1 (see Note 8).
- The concentration of MgCl2 may be altered (0.5–5 mM) to optimize the specificity and yield of the reaction.
- The design of the ASO PCR primer is essential for specific amplification of the template. Primers are synthesized in two forms (the wild or normal type and the mutant), with the correspondingly different bases at the 3′ end. However, a single mismatch is often not enough to prevent nonspecific amplification and the introduction of additional deliberate mismatches near the 3′ terminal end (e.g., four bases from the 3′ end) of the primers may overcome this problem. Several investigations have examined the effect of the type of the 3′ terminal primer-template mismatches on the PCR amplification, however, it appears to differ depending on the gene being studied. Where possible, select primers of random-based distribution and approx 50% GC content. The primers should not be complementary to each other or contain a sequence with significant secondary structure. The common primer should be designed to give a product of suitable size (e.g., 200 bp).
- A concentration 100 ng of template DNA is usually sufficient to amplify.
- Two reactions are required for the detection of a point mutation: one including the wild-type and common primers, and the other with the mutant type and common primers.
- Addition of the Taq polymerase following the initial denaturation step while the PCR reaction is held at 80°C prior to the cycling reactions may increase the specificity of the PCR products.
- The volume of PCR reaction can be altered according to requirements: 10–100 μL.
- These are standard PCR reaction conditions, which may not amplify the template specifically. By varying the conditions and constituents of the reaction (altering the magnesium [0.5–5 mM], deoxynucleotide triphosphates [dNTPs] [50–200 μM], ASO primer concentration, DNA template, and Taq polymerase concentration, and increasing the annealing temperature), this may be overcome. A good indication of the correct annealing temperature is the melting template of the oligonucleotide primers. This can calculated using the formula 64.9 + 0.41 (%C + %G) –600/n. The addition of specificity enhancers such as DMSO (10%), may also increase the specificity. However, it may be necessary to redesign the primers altering the 3′ mismatches.
- Amplification of a control DNA template with known point mutations will aid the establishment of the ASO PCR. By including an internal control reaction, such as β-globin amplification, the risk of false negatives will be reduced.
In this application, three nucleotides are studied. These nucleotides sequences are complementary to the sense strand of the normal human b- globin gene. A substitution in the nucleotide sequence coding for codon 6 is the difference between the nucleotide sequences. The oligodeoxynucleotides are complementary to the genes in the region of the mutations and are therefore allele-specific. When radiolabeled and used as hybridization probes, the oligodeoxynucleotides are found to hybridize specifically to the mRNA transcribed from each allele.
MATERIALS AND METHODS
(a) RNA preparation
RNA was prepared from 20 ml of heparinized blood obtained from various individuals of differing B-globin genotypes. Red cells were lysed by resuspending the washed cell pellet in 0.144 M NH4Cl, 3 mM DTT followed by the addition of 0.1 vol. of 0.01 M NH4HC03. To the lysate is added 0.1 vol. of 1.5 M sucrose, 0.5 M KCl and 0.5% SDS and the solution was centrifuged at 3000 x g for 20 min at 4°C. The ribonucleoprotein was recovered from the supernatant by precipitation at pH 5 using 10% acetic acid followed by centrifugation at 3000 x g for 20 min at 4°C. The pellet was dissolved in 0.1 M Tris. HCl pH 9, 0.1 M NaCl, 1 mM EDTA and 0.1% SDS and extracted first with phenol-chloroform and then with chloroform-isoamyl alcohol followed by ethanol precipitation.
(b) Gel electrophoresis and blotting
RNA was denatured by dissolving in 6 M glyoxal and 50% dimethyl sulfoxide and heating at 50oC for 60 min. The denatured RNA was subjected to electrophoresis on a 1.5% agarose gel using 0.01 M sodium phosphate pH 7 buffer. Electrophoresis was at 3.5 V/cm for 3 h. RNA was transferred to hybridization chamber by capillary blotting using 20 x SSC buffer. The blots were dried at room temperature and baked in vacua at 80°C for 2 h.
(c) Probe labeling
The three 19-nt oligos Hb19A’, Hb19S’ and Hb19C’ were synthesized on an automated DNA synthesizer. The oligos were radiolabeled at their 5’-ends using ( g-32P) ATP and T4 polynucleotide kinase and purified by electrophoresis on a 19.35% acrylamide, 0.65% bisacrylamide, 7 M urea gel.
The blots were prehybridized in 10 x Denhardt’s solution (0.2% bovine serum albumin, 0.2% polyvinylpyrrolidone and 0.2% Ficoll) and 0.1% SDS for 1 h at 60°C. The blots were then washed in 2 x SSC and hybridized with the labeled oligo (1 x 106 cpm/ml) in 5 x Denhardt’s, 5 x SSPE and 0.1% SDS for 3 h at 60°C (58°C in the case of H/319C’). For the hybridizations where unlabeled competitor oligo is included, it is added in a 10-fold molar excess over the labeled one. After hybridization, the filters were washed three times with 6 x SSC at room temperature for 15 min followed by one wash with 6 x SSC at 57°C for one min. The filter is then exposed to X-ray film with two x-ray image intensifier screens at -70°C for 0.5-3 h.
The position and length of the sequences are based on several considerations:
(1) The length of 19 nt has shown to give a probe that recognizes a unique sequence in the human genome.
(2) The mismatches are centrally located to optimize thermal destabilization.
(3) All sequences are anti-sense and are thus complementary to the mRNA.
(4) Each oligo is complementary to one allele of the B-globin gene and forms either one or two mismatches with the other alleles. Hb19A’ is specific for the bA allele, Hb19S for the bs allele and Hb19C’ for the bC allele. For simplicity of discussion, an oligo which forms a duplex with no mismatches will be called c-oligo (complementary oligo) and one capable of forming duplexes with one or more mismatches will be termed non-c-oligo.
Effect of G: T mismatch
Hybridization of a c-oligo to its target sequence is unaffected by the presence of a molar excess of unlabeled non-c-oligo. Therefore, to decrease the hybridization of non-c-oligos forming a G: T mismatch to the noncomplementary target sequence, hybridizations were attempted using labeled c-oligo in the presence of a lo-fold molar excess of unlabeled non-c-oligo. The presence of the competitor oligo should effectively suppress any hybridization of the labeled oligo to its noncomplement~ target sequence.
mRNA was isolated from blood cells of individuals which were either homozygous for the normal b-globin gene (AA) or heterozygous for the normal and bC allele (AC). The RNA was glyoxylated in the presence of DMSO, subjected to electrophoresis on an agarose gel and transferred to GeneScreen. Duplicate Northern blots containing the two RNAs were hybridized with [32P]Hb19C’ in the absence or presence of a 10-fold molar excess of unlabeled Hb19A’. In both cases, the Hb19C’ probe hybridizes strongly with the b-globin mRNA present in the AC RNA. In the absence of the competitor, however, there was a low level of binding of the Hb19C’ probe to the normal b-globin mRNA in the AA sample. This residual hybridization could only be reduced by long high-criteria washes (65°C in 6 x SSC) which also resulted in the loss of signal from the AC lane. In the presence of the non-c-oligo competitor, there is essentially no binding of the Hbl9C’ oligo to the bA-globin mRNA.
Specific hybridization of allele-specific probes to bA, band bc mRNA
RNA was isolated from the blood cells of individuals with the following b-globin genotypes: AA, AC, AS and SS. The total RNA was denatured, subjected to electrophoresis and blotted onto a GeneScreen filter. The blot containing the four RNA samples was hybridized first with Hb19A’ [32P]probe in the absence of unlabeled non-c-oligo. After the hybridized probe was removed, the filter was rehybridized with Hb19S’ [32P]probe in the presence of unlabeled Hb19A’ oligo. The hybridized probe was once more removed and the filter was again rehybridized with Hb19C’ [32P]probe in the presence of unlabeled Hbl9A’ oligo. As can be seen, each probe only hybridized to RNA samples containing the homologous fl-globin mRNA and not to samples cont~ing non-homologous allelic transcripts. Thus, the allele-specific oligo probe can obviously astonish among the transcripts of allelic genes both in the case where the genes differ by a single transversion mutation as for bA vs bs as well as in the case where the genes differ by a single transition mutation as for bA vs bc.
Under appropriate conditions, oligos will only form duplexes with complementary DNA sequences when perfect base pairing is possible. All non- Watson/Crick bp have a destabilizing effect. Thus, oligo probes are capable of discriminating between alleles which differ by as little as a single bp. This allelic hybridization specificity was first demonstrated in experiments designed to determine carriers of the genetic disorder, sickle-cell anemia. 19-bp oligos were used to discriminate the wild-type b-globin allele (bA) from the mutant bc allele, which is responsible for sickle-cell anemia in the homozygous state. Even though these alleles differ by a single bp, oligo hybridization unambiguously detected each gene when present in either homozygous or hemizygous states. Similar discrimination has been demonstrated for normal and mutant genes in several other genetic diseases. Obviously, it is highly desirable to be able to discriminate between RNAs which differ by as little as a single nt. In this paper, we show that this is indeed possible, even in the case where the probe forms a single G: T mismatch with the noncomplementary transcript. This is accomplished by having an excess of unlabeled non-c-oligo present during the hybridization with 32P-labeled c-oligo. The non-coligo blocks hybridization of the labeled c-oligo sequence to the non-c sequence without affecting hybridization of the labeled c-oligo to the complementary sequence. Use of the hybridization conditions described here should enable one to examine the transcription of highly related genes present within the same cell, either allele differing by one or more bp [e.g., the alleles of the H-ras or N-rus genes or nonallelic, but highly homologous genes (e.g. MHC class I genes]. It should also be possible to quantitate such transcripts through the use of an appropriate internal control. The competition hybridization technique should also be useful in oligo hybridization to DNA sequences which differ by only a single nt.