Week 3 - L6 - PCR-Based Identification of Allelic Variants and Tandem Repeats

The identification of allelic variants can be achieved through tests based on the products generated from the Polymerase Chain Reaction (PCR) amplification of specific genes.
PCR functions by utilizing specific primers that bind to a target region of interest.
Through repeated cycles of amplification, PCR produces a specific product corresponding to that target region.
To identify different types of genomic variants, specific steps of the PCR process can be manipulated or utilized, particularly the primer annealing and the resulting product size.

One primary method for identifying variants involves altering the conditions or components of the annealing step.
Primers can be designed so they only anneal to the template if a specific variant is present.
If the variant is absent, the primer fails to work, effectively stopping the production of the PCR product. Conversely, the presence of the variant allows for primer binding and successful amplification.
This technique is highly effective for identifying single nucleotide variants ( $SNVs$ ) and insertions/deletions ( $indels$ ), as these mutations significantly impact the ability of a primer to anneal to its intended target.

ARMS PCR is a specific technique used to genotype a particular locus based on whether primers can bind to specific variants.
The design of ARMS PCR primers focuses on the $3'$ end of the primer sequence.
The $3'$ end is the most critical part of the primer because DNA synthesis begins at this point on the template strand.
If the $3'$ end does not bind perfectly to the template, extension of the product is highly unlikely or will not occur at all.

Outer Primers: These are a pair of primers (one forward, one reverse) that flank the entire region of interest. They are designed to always produce a product regardless of the internal variant present. This serves as a positive control to confirm that the PCR reaction actually worked.
Internal Variant-Specific Primers: Two additional primers are added, each specific for a different allele at the variant site.
Allele-Specific Binding Scenarios:
- In an example involving a $G/T$ variant:
  - A primer with a $G$ at its $3'$ end will be complementary to a $C$ on the template (corresponding to the $G$ allele locus). When this match occurs, it fires toward the opposite outer primer to create a specific, smaller product.
  - If the template contains a $G$ but the primer provided has an $A$ at the $3'$ end, no binding occurs because $A$ is not complementary to $G$ , and no product is made.
  - A primer with an $A$ at its $3'$ end is specific for the $T$ allele (complementary to the $T$ on the template). It will fire in the opposite direction toward the other outer primer, creating a distinct product.
  - If the template contains a $T$ but the primer provided has a $G$ at the $3'$ end, no binding occurs because $G$ is not complementary to $T$ .

Common Amplicon: All samples should show a large band representing the product formed by the two outer primers.
Homozygous for Allele 1 (e.g., $G$ ): Shows two bands—one large common amplicon and one smaller product generated by the $G$ -specific inner primer.
Homozygous for Allele 2 (e.g., $A/T$ ): Shows two bands—the common amplicon and a product generated by the $A/T$ -specific inner primer. This band will typically differ in size from the $G$ product if the inner primers are positioned at different distances from the outer primers.
Heterozygous: Shows three bands—the common amplicon, the product for the first allele, and the product for the second allele.

Variant identification can also rely on differences in the size of the PCR product. This occurs when a variant changes the physical length of a genomic region.
This is specifically applicable to repeat sequences known as micro and mini satellites, which are distributed widely across the genome.
General Characteristics:
- These are short sequences repeated in tandem.
- They are often located in regions with no impact on gene function but can occasionally be associated with genetic conditions.
- They are highly polymorphic. Unlike $SNVs$ which have a maximum of four options ( $A, T, G, C$ ), repeat sequences can have a vast range of variants (e.g., $4, 5, 6, 7$ up to $50$ or more repeats).
- Due to this high variation, they are primary tools for DNA fingerprinting in forensic science and are used to distinguish between individuals.
- Individuals are highly likely to be heterozygous at these locations, possessing different numbers of repeats on each homologous chromosome.

Microsatellites (Short Tandem Repeats or STRs):
- Composed of very short repeat units, typically $2\text{ to }10\text{ base pairs}$ ( $bp$ ).
- Example: Repeating units like $AT$ , $ATT$ , or $ATT$ .
Minisatellites (Variable Number of Tandem Repeats or VNTRs):
- Larger repeat units, generally between $10\text{ to }100\text{ base pairs}$ ( $bp$ ).
- These can be repeated from a few times to $N$ number of times.

Detection is based on the principle: more repeats equals a larger PCR product.
Procedure:
- Primers are designed to bind to the unique regions on either side of the tandem repeat.
- If an individual has one allele with $4$ repeats and another with $8$ repeats, the PCR will produce two different fragments.
- The fragment with $4$ repeats is smaller and will migrate faster through a gel during electrophoresis.
- The fragment with $8$ repeats is larger and will migrate more slowly.
The resulting band pattern tells the researcher the genotype (the specific number of repeats) for that individual at that specific locus.

Repeat regions serve as molecular markers to tell chromosomes apart.
Molecular Mapping Example:
- Consider a pedigree where a female has alleles with $3$ repeats and $5$ repeats, and a male has alleles with $2$ repeats and $8$ repeats.
- These repeats serve as identifiers ( $M', M'', M''', M''''$ ) for specific chromosomes.
- If a pathogenic variant is in linkage with a specific marker (e.g., a dominant disease allele is physically located next to the $5$ -repeat marker on the same chromosome), we can track the inheritance of the disease by tracking the marker.
Case Study Analysis:
- In an affected family, all individuals with a specific dominant condition also carry the $M''$ marker ( $5$ repeats).
- Unaffected individuals do not carry this marker, instead possessing markers in linkage with the recessive, non-pathogenic allele.
- This demonstrates that the pathogenic variant is located in the vicinity of that specific molecular marker.
Clinical Use:
- This knowledge can be applied to future offspring through methods like non-invasive prenatal testing ( $NIPT$ ).
- By identifying whether a fetus has inherited a specific marker ( $M''$ ), clinicians can determine the high likelihood of the child inheriting the associated pathogenic allele.