"A method used to detect a single base change or SNP using sequence-specific primers is called ARMS or allele-specific PCR."
The PCR technique can detect mutations such as deletion, duplication, insertion, or single base change of SNPs. It is a temperature-dependent amplification technique based onTaq-DNA polymerase.
There are many variations of PCR depending on the requirements of the assay,ARMS-PCRIt is one of them. It goes by other names like allele-specific PCR, PASA, or AS-PCR, all of which have similar uses.
Please let us know the full names, the initials ARMS asAreinforcementRundisciplinedMETROutationSSystem. acronym PASS asPAGRCAreinforcement ofSspecificAmean and AS-PCRAAlto-Sspecific PCR.It often uses the word "allele" because of its specific amplification ability for a particular allele or DNA sequence.
kary bubbledescribed the technique ofin vitroReinforcement in 1983. After several years discovering the truePCR-Technik,CR Newtonand his coworkers discovered the ARMS-PCR, or allele-specific PCR technique.
The present technique has applications in the detectionSNP (single nucleotide polymorphism)and genotyping. That is all we will discuss in this article.
This article provides information on ARMS-PCR, its principle, process, protocol, and steps. I also explained the advantages, disadvantages and applications.This article will increase your PCR knowledge and will certainly help you in your studies.
Stay tuned.
Key topics:
What is ARMS or allele-specific PCR?
Alleles are alternative forms of genes. When a mutation occurs, it produces two different alleles for a gene; one mutant and one normal. Allele-specific PCR has the ability to detect a single specific allele.
That is, if you only want to amplify one mutant allele, create a suitable primer set and amplify it using this technique. Each specific primer set is designed for each specific allele.
However, we need to make a small change to the primers to amplify each allele. Subsequently, PCR amplifies each allele simultaneously. While gel electrophoresis prepares the results for evaluation.
Two different sets of primers for separate alleles are prepared. Mutant allele-specific primers are refractory (resistant) to normal alleles and vice versa. Hereinafter known as the Amplification Refractory Mutation System.R. Newton called it ARMS-PCR.
Principle of ARMS-PCR:
The principle of the present technique is based on the modification of primers to amplify a specific allele.
The 3' end of the primer is modified so that one primer can amplify a mutant allele while the other can amplify the normal allele. more researchersmodify some bases of the 3'-OH end of the primer.
Only a single set of primers can amplify only one specific allele present in a sample during the reaction. Gel electrophoresis helps verify results.
The concept of incompatibility:
By introducing a mismatch into a primer, refractory amplification is allowed. Here, the mismatch between the primer and the DNA template plays a crucial role in achieving amplification.
The mismatch actually changes the annealing temperature for different sets of primers. Here are examples of various incompatibilities.
Disco de Starke: G/A, C/T, T/T
Average deviation: A/A, G/G, C/C,
Weak discrepancy: C/A, G/T
Think about this question, I will answer it later. Why don't we use high fidelity Taq DNA polymerase in ARMS-PCR?
See the image below so that you can easily understand the concept.
ARMS-PCR procedure and steps:
The procedure is simple and effective. Dangerous radioactive probes are not involved here. We can divide the whole process into 4 different steps:
- First-Design
- reinforcement
- Agarose-Geleeletrophorese
- interpretations of the results
First-design:
The preparation and design of the primer play a decisive role here. Remember that the primer must be allele specific. Some points to keep in mind when preparing are:
First look at the photo above. Suppose our DNA sequence has a G-A point mutation, that is, G in the normal allele and A instead of G in the mutant allele.
We need to design a forward primer such that, for the normal allele, the primer contains C (complementary to G) at the 3' end and the mutated primer contains T instead of C.
Although it works magically if we add a mismatched base near our SNP at the 3' end. You may be wondering why we need to add a discrepancy. The mismatch is the key factor in achieving the gain.
A weak mismatch increases the probability of amplification, so add a strong mismatch to avoid false amplification near the 3'-OH end of the primer (ideally at position -2). The mismatch prevents binding to the non-complementary sequence and terminates the amplification.
C:T, G:A and A:G are the severely mismatched base pairs that slow the amplification process up to 100 times.We only need to modify a single primer, either forward or backward, the other primer is usually unchanged and can work for either allele.
Additionally, the primer must meet all standard primer design criteria. It should have less GC content, less hairpin structure, and be between 20 and 30 nucleotides in length.
Reinforcement:
We usually don't discuss the amplification process separately. but to getcorrect' Amplification, we need to maintain healthy amplification conditions. here are some
Set a higher annealing temperature based on the highest annealing temperature of the primer. Setting it lower increases the chance of non-specific amplification, remember we're only playing with a single base switch initiator.
Prepare the reaction according to the following table.
Set a lower PCR cycle to maintain the specificity of the reaction. The ideal is to program only 22 to 25 cycles. More PCR cycles increase the probability of nonspecific binding, primer dimer, and redundant results.There are false positive results.
"An additional mismatch at the 3' end after the first mismatch increases the specificity of the ARMS-PCR reaction."
Use some built-in controls to monitor the results. Include positive, negative, and internal control tubes in the reaction.
Agarose gel electrophoresis:
ARMS-PCR has direct diagnostic utility as it does not require any hybridization steps.Results are measured on a 2% agarose gel, ideally under the proper running conditions of the agarose gel. Add along with a 1000 bp molecular marker.
OTechnik der Gelelektrophoreseit is enough to separate different alleles and evaluate the results. The ARMS PCR results are interesting, go to the next section.
Results and interpretation:
To verify the results, the first step is to observe the internal control. The internal control band must be present in all reactions. This shows that our reaction setup, cycle conditions, and other practices are in order.
We show the results in the gel image provided above. Check it out first. Now analyze each band for normal and mutant alleles. To understand the results, let's set up a hypothetical situation.
Suppose we have three samples; one normal, one heterozygous carrier, and one homozygous dominant carrier. We prepared 2 tubes for each sample, totaling 6 tubes for samples with a positive control and two negative controls.
see illustration
As shown in the figure above, homozygous normals show a single DNA band, heterozygous carriers show two bands (one for the normal allele and one for the mutant allele), and homozygous disease states show a single mutant band.
It can also be said like this, the mutant primer cannot amplify the normal homozygous allele, both primers amplify both alleles in the heterozygous state, and only one mutant primer can amplify the mutant allele.
Advantages of ARMS-PCR:
Each PCR variant has its own meaning, just like allele-specific PCR. Here I have listed some of its advantages.
- The technique can accurately distinguish two different alleles.
- It can recognize a single base variation - SNP.
- It is a precise, fast and reliable PCR technique.
Limitations of ARMS-PCR:
- It includes a complex primer design and incompatible embedding techniques.
- It can only recognize a few SNPs at a time.
- You can accurately distinguish between homozygous and heterozygous alleles.
- It is one of the most important techniques for genotyping and allelic variation studies.
- It contains more primer sets and is therefore an expensive process.
- It can only recognize known SNPs, new variations or mutations cannot be recognized.
- There is a higher chance of false positive results and therefore negative and positive internal controls are required each time.
- It is temperature sensitive, which means that a small change in annealing temperature will cause a serious problem in the reaction.
- Finally, like other PCR variants, it cannot examine chromosomal changes, major mutations such as deletions and duplications.
ARMS-PCR applications:
- The ARMS-PCR technique is used exclusively for SNP detection, genotyping and discovery of allelic variations.
- It is often used to detect various mutations.
- It also has uses in diagnosis. For example, it recognizes different beta thalassemia alleles or mutations using sequence-specific primer sets. It is a gold standard method for detecting mutations associated with beta thalassemia and sickle cell disease.
- Investigators also use allele-specific PCR to detect JAK2 mutations and HIV variants. Commercial kits are now available for many assays.Knock and others. used this technique in genotyping HIV-1 for ATT gene deficiency. Read his research here,Click here.
Optimization for ARMS-PCR:
Our blog is not just for collecting and providing information that is available on the internet. We also share our own experiences. I have great experience in PCR technology and that is why we add more value to the subject.
In this section, I will give some optimization tips that will help not only students, but also new researchers in their experiments. Here are my suggestions
- Avoid longer primers for ARMS-PCR, use a primer length between 25 and 28 nucleotides.
- Incorporate a single mismatch in the second position at the 3-OH end.
- Add a weak second mismatch in third position.
- Use 10 pM each ARMS primer, use 1 to 3 pM internal primer.
- Generally, use shorter PCR programs between 25 and 28.
- Use MgCl2 for the reaction to increase the amplification specificity. Ideally, use > 1.5 mM.
Do you remember the question I asked at the beginning? Why don't we use high fidelity DNA polymerase in ARMS-PCR? Here is the answer,
In conventional ARMS-PCR, we only use a single Taq DNA polymerase. High fidelity DNA polymerase has polymerase and exonuclease activity and therefore eliminates mismatches.
Interesting fact: The conversion of "guanine" to other nucleotides is more common than the rest.
Variants of ARMS-PCR:
Scientists are modifying the native ARMS-PCR technique to perform different functions. These are some of the modifications.
Concept of T-ARMS-PCR:
T-ARMS-PCR, called tetra-primer-PCR or tetra-ARMS-PCR, is used to detect various mutations and therefore has great diagnostic value. It uses 4 different primers for two separate alleles of a gene.
This means one set of forward and reverse primers for a normal allele and another set of forward and reverse primers for a mutant allele.
Multiplex-ARMS-PCR:
Multiplex ARMS-PCR is used for a similar purpose but can save experimentation time and cost. Here two separate multiplex reactions were performed for more than two SNP detections.
It is more complex and increases the possibility of erroneous results. Considerable experience in the relevant field is required to design the trial and run the results.
qARMS-PCR:
It is a technique that not only amplifies alleles, but also quantifies the specific allele. From now on, it can tell us the number of normal or mutant alleles in the sample.
Diploma:
To understand the concept of allele-specific PCR or ARMS-PCR, one must have a strong experimental background. At least they know different types. It is very difficult for a newbie or student to understand the concept.
Because the concept is a bit complicated. We make serious changes to the primers, they are generally not recommended.
The technique has great utility in SNP detection, genotyping and mutation and I personally developed and used it during my research time.
Trust me, it was fun to do trial and error. I hope you like this article. if so pleasebrand pagesin your browser.
Fuentes:
Darawi, MN, Ai-Vyrn, C., Ramasamy, K.and anotherAllele-specific polymerase chain reaction to detect Alzheimer's-related single nucleotide polymorphisms.BMC Med Genet14, 27 (2013).https://doi.org/10.1186/1471-2350-14-27.
Gaudet M, Fara AG, Beritognolo I, Sabatti M. Allele-specific PCR in SNP genotyping. Methods Mol. Biol. Rev. 2009;578:415–2 doi: 10.1007/978-1-60327-411-1_26. PMID: 19768609.