Allele-Specific Amplification (ASA) by Amplification Refractory Mutation System (ARMS) PCR
Inquiry NowAllele-specific amplification (ASA) by a refractory mutation system (ARMS) PCR is a PCR-based method that can be used to detect known single nucleotide polymorphisms (SNPs). ASA uses two pairs of primers in a single PCR reaction: one pair of non-allele-specific external primers and the other pair of allele-specific internal primers. The 3' end of the internal primers is fully complementary to the wild-type or variant sequence, while the external primers are irrelevant to the target sequence. If the internal primer does not match the template sequence, amplification will be blocked, so only the internal primer that matches the template sequence can produce a specific product.
ASA is also known as ARMS or PCR amplification of specific alleles (PASA) and was first established by Newton et al. to detect known mutations. ASA has high sensitivity and specificity and can be analyzed on conventional PCR and electrophoresis instruments without the use of expensive fluorescent probes or restriction enzymes. ASA can detect multiple SNPs simultaneously and can also be combined with real-time PCR technology for quantitative analysis.
ASA has important applications in molecular diagnosis and gene therapy. ASA can be used to detect SNPs related to human genetic diseases, infectious diseases, tumor markers, etc., providing a basis for clinical diagnosis, prognosis assessment, drug treatment, and genetic counseling. ASA can also be used to evaluate the effects of gene therapy, such as monitoring the expression and integration of target genes in transfected cells.
Steps of ASA by ARMS PCR
The experimental steps of ASA by ARMS PCR include sample preparation, DNA extraction, PCR amplification, electrophoresis detection, etc.
Table 1. Experimental steps of ASA by ARMS PCR
| Step | Description | Parameters/Notes |
|---|---|---|
| Sample preparation | Obtain DNA samples from blood, saliva, tissue, or other sources, or use commercial DNA standards. | Sample quality and quantity should be sufficient to avoid contamination and degradation. |
| DNA extraction | Extract DNA using the phenol-chloroform method, column chromatography method, or other methods, and measure concentration and purity. | DNA concentration should be between 50–100 ng/μL, A260/A280 ratio should be between 1.8–2.0. |
| Primer design | Design two pairs of primers based on the target sequence, one pair of outer primers and one pair of inner primers. Outer primers are used to amplify the region containing the SNP, inner primers are used to distinguish different alleles. | Primer length should be between 18–30 bases, Tm value should be between 55–65°C, GC content should be between 40–60%. Avoid secondary structure and dimer formation. The 3' end of the inner primer should match the target SNP perfectly, and the 3' end of the outer primer should have at least one base mismatch with the inner primer. |
| PCR amplification | Add the DNA template, four primers, dNTPs, MgCl2, buffer, and Taq DNA polymerase to a single reaction system and perform PCR amplification. | Reaction volume is usually 25 μL, each primer concentration is usually 0.1–0.5 μM, dNTPs concentration is usually 200 μM, MgCl2 concentration is usually 1.5–2.5 mM, buffer pH is usually 8.3–8.5, and Taq DNA polymerase concentration is usually 1-2 U. The PCR program is usually: initial denaturation at 94°C for 5 min; 35 cycles: denaturation at 94°C for 30 s; annealing at 55-60°C for 30 s; extension 72°C for 30 s; final extension 72°C for 10 min; 4°C storage. |
| Electrophoresis detection | Separate PCR products by agarose gel electrophoresis and visualize with ethidium bromide staining. Determine genotype based on product band number and size. | Agarose gel concentration is usually 2–3%, voltage is usually 100–120 V, and electrophoresis time is usually 30–60 min. Product band number and size are related to the distance between inner and outer primers; genotype can be determined according to the following rules: If there are only two bands (product between outer primers and product between one of the inner primers and outer primer), it is homozygous; if there are three bands (product between outer primers and products between both inner primers and outer primer), it is heterozygous; if there are no bands, it is negative control or reaction failure. |
Applications of ASA and ARMS PCR
ASA and ARMS PCR are useful techniques for the detection of single nucleotide polymorphisms (SNPs), which are the most common type of genetic variation in human beings. SNPs can affect the function of genes and proteins and are associated with various diseases, traits, and responses to drugs. Therefore, ASA and ARMS PCR have wide applications in clinical diagnosis, genetic screening, pharmacogenetics, and population genetics.
In clinical diagnosis, ASA and ARMS PCR can be used to detect SNPs that cause or predispose to certain diseases, such as Alzheimer's disease, cystic fibrosis, thalassemia, galactosemia, hypertension, diabetes, and mitochondrial disorders. The detection of these SNPs can help in the diagnosis, prognosis, risk assessment, and treatment of patients.
In genetic screening, ASA and ARMS PCR can be used to identify carriers or affected individuals of certain genetic disorders in families or populations. For example, ASA and ARMS PCR can screen for SNPs that cause hemophilia A, Duchenne muscular dystrophy, fragile X syndrome, and Huntington's disease. These SNPs are located in genes such as F8, DMD, FMR1, and HTT, which are involved in these disorders. The screening of these SNPs can help in genetic counseling, prenatal diagnosis, and prevention of these disorders.
In pharmacogenetics, ASA and ARMS PCR can be used to detect SNPs that influence the metabolism, efficacy, and toxicity of drugs. For example, ASA and ARMS PCR can detect SNPs in genes involved in the metabolism or action of drugs such as codeine, warfarin, azathioprine, and methotrexate23. The detection of these SNPs can help in personalized medicine, dose adjustment, and adverse reaction monitoring of these drugs.
In population genetics, ASA and ARMS PCR can be used to study the genetic diversity, evolution, and migration of human populations. For example, ASA and ARMS PCR can detect SNPs in genes related to the immune system, paternal lineage, maternal lineage, and skin color of human populations. The detection of these SNPs can help in the understanding of the origin, history, and adaptation of human populations.
