What is the ARMS-PCR technique?
Nowadays, a wide range of molecular techniques have been developed in order to identify genetic mutations like Sanger sequencing, which unfortunately does not have the capability to efficiently screen a large number of mutations in samples. However, this limitation can be overcome by employing the next generation sequencing (NGS) approach. It is important to note that both Sanger sequencing and NGS methods tend to be quite costly. On the other hand, a simpler and more economical alternative known as ARMS-PCR, or tetra-primer amplification refractory mutation system-polymerase chain technique, can be employed to ascertain the genotype of single nucleotide polymorphisms (SNPs).
This method is considered to be one of the most precise diagnostic tools for genetic diseases and is currently recognized as the gold standard for diagnosing thalassemia and sickle cell anemia. The ARMS-PCR method operates by utilizing sequence-specific PCR primers that will only be amplified if the target nucleotide sequence is present in the DNA sample. Once the ARMS reaction has taken place, the presence or absence of the PCR product is utilized to diagnose the presence or absence of the target allele.
In this method, two primers are utilized with an allele-specific oligonucleotide primer located at the 5′ end and a common primer located at the 3′ end. If the primer designed for the mutant allele is successfully amplified, resulting in a visible band on the electrophoresis gel, it indicates that the target sequence contains the mutant allele. Conversely, if the primer designed for the mutant allele fails to amplify, it suggests the presence of a normal DNA sequence at that specific point.
Primers applied in the ARMS-PCR approach
Within the ARMS-PCR technique a set of primers is utilized, including both forward and reverse primers. Two distinct forward primers are designed for two mutated and natural alleles, ensuring that their 3′ ends align with the desired nucleotides. In primer design, emphasizes the significance of complementarity and pairing of the 3′ end nucleotide in primers. If this particular base fails to pair with its complementary base, the amplification of the fragment will not occur and leading to the absence of a visible band during gel electrophoresis. Conversely, the reverse primer complements the allele sequence and remains unchanged. Consequently, this technique is currently employed for the identification of point mutations or SNPs.
The number of cycles in this technique is fewer in comparison to conventional PCR, typically ranging from 20 to 22 cycles. Given that the ARMS-PCR technique is predominantly utilized for the detection of mutations or polymorphisms, it is crucial to ascertain whether an allele is homozygous or heterozygous through the use of primers. In certain cases, normal and mutated primers are placed in separate microtubes, and their PCR reactions are conducted independently. However, if the normal and mutated primers are labeled using fluorescent dyes, there is no need to separate the reactions.
One of the commonly types of the ARMS-PCR technique is the Tetra ARMS-PCR method, in which the number of primers employed differs. In this particular approach, the amplification of the target allele is executed with the aid of four primers, encompassing two inner primers and two outer primers. The outer primers fulfill a similar function as the primer utilized in traditional PCR and contribute to the amplification of the desired DNA sequence. Conversely, the internal primer is employed to ascertain allelic mutations, such as the presence of Single Nucleotide Polymorphism (SNP). The Tetra ARMS PCR reaction is conducted within a single tube and a solitary PCR step, thereby enabling direct observation of genotypic changes of target SNPs through conventional agarose gel electrophoresis.
Advantages of ARMS-PCR technique
- Detection of different SNPs associated with diseases
- The most reliable method in diagnosing thalassemia and sickle cell anemia
- Short duration of the reaction
- No need to use restriction enzymes in polymorphism detection
- Determining whether a person is heterozygous or homozygous in a gene locus
- Identification of JAK2 and HIV mutations
Limitations of the ARMS-PCR technique
- Limitation in the detection of chromosomal abnormalities
- Non-recognition of deletions and duplications
References
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9558987/
https://journal.waocp.org/article_32475_b4309ff77054c351a1f76c13d7bbb04b.pdf
https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-018-3236-6
