Gel Electrophoresis and Genetic Analysis

DNA is a large molecule comprised of a sequence of four nucleotides: adenine (A), cytosine (C), thymine (T), and guanine (G).
Humans have 46 DNA molecules organized into 46 chromosomes.
Each chromosome contains thousands of genes that code for various traits like hair color, eye color, and the presence of genetic diseases.
Genetic diseases can result from mutations in a single gene.

Gel electrophoresis is a technique used to identify specific genes in the DNA.
It involves cutting DNA into smaller fragments using restriction enzymes.
Restriction enzymes break DNA at specific nucleotide sequences.
DNA fragments are placed into wells in an agarose gel (a semi-solid substance with microscopic catacomb-like structures filled with water).
An electric current is applied to the gel, with a negative charge near the wells and a positive charge at the far end.
The negatively charged DNA fragments are pulled through the gel towards the positive end.
Smaller DNA fragments move more quickly through the gel than larger fragments.

Restriction enzymes are crucial for identifying mutations.
For example, the restriction enzyme EcoR1 binds to every GAA TTC sequence in the DNA and cuts between the G and the first A.
If a DNA sequence contains multiple EcoR1 binding sites, the enzyme will cut the DNA at each site, resulting in multiple fragments.
In gel electrophoresis, each DNA fragment appears as a band, indicating the presence of restriction enzyme sites in the DNA.
Restriction enzymes can cut any DNA as long as their specific binding site is present.

The way DNA is cut by restriction enzymes varies among individuals, leading to variations in the length of the resulting fragments.
Variation is referred to as polymorphism.
Restriction Fragment Length Polymorphisms (RFLPs) are variations in the length of DNA fragments after digestion with restriction enzymes.
If a polymorphic sequence is also a restriction enzyme binding site, it is called a polymorphic marker.
Polymorphic markers identify specific spots in the DNA where polymorphisms can be identified using restriction enzymes.
If there is a mutation in a restriction enzyme binding site the enzyme will not be able to bind and cut at that location.

A Single Nucleotide Polymorphism (SNP) is a mutation in a single nucleotide.
If an SNP occurs in a restriction enzyme binding site, the restriction enzyme may no longer be able to cut the DNA at that site.
This changes the size and number of DNA fragments produced after restriction enzyme digestion.
Gel electrophoresis can then be used to identify the mutation based on the altered fragment pattern.

A Short Tandem Repeat Polymorphism (STRP), also called a microsatellite, is a repeating DNA motif (e.g., CAT) repeated multiple times (5-50+ times).
The repetitiveness of STRPs can cause issues during DNA replication, as DNA polymerase can slip and replicate the repeat extra or fewer times, leading to variations in allele length compared to the original.
If a restriction enzyme cuts DNA containing STRPs, fragments with more repeats will be longer than those with fewer repeats.
Gel electrophoresis can track these size differences.

Gel electrophoresis helps visualize DNA mutations and repeats, which can be used as markers to track genes of interest.
Human chromosomes are organized into 23 pairs of homologous chromosomes.
During gamete formation, homologous chromosomes exchange genetic material through a process called crossing over.
Crossing over involves the twisting and exchange of parts between the ends of paired chromosomes, creating genetic variation.
However, crossing over can make it difficult to track genes that move from one chromosome to another.
Genetic linkage states that the closer two genes are located on a chromosome, the less likely they are to separate during crossing over.
If a marker and a mutant gene are close to each other, they are unlikely to separate during crossing over.

If a known point mutation or SNP is located near or within a gene, identifying individuals with the SNP using gel electrophoresis suggests they likely have the gene of interest.
To utilize gel electrophoresis, it's essential to determine how close a marker is to the gene of interest.
One method involves studying a family pedigree with individuals affected by the disease.
Test all family members for available markers and correlate their presence with the appearance of the disease.
For instance, if four markers (SNPs) are tested, and marker two is found in all seven diseased individuals but also in two healthy family members, it indicates that the marker is close to the disease gene but not perfectly linked.
The two healthy individuals inherited the SNP but not the mutant allele, suggesting that crossing over separated the marker and the mutant allele in 2 out of 9 divisions.
In practice, affected pedigrees are typed for hundreds of markers across the entire genome to select the closest and most reliable marker.

Genetic testing employs various methods to identify the mutant allele causing a disease.
The mutant allele is tracked down and reliably identified using markers that follow it through crossing over events in meiosis.
Marker candidates include SNPs and STRPs.
A pedigree is created for the affected family, and members are analyzed for the most reliable marker.
The reliability of the marker indicates its proximity to the disease gene, with closer markers being more effective.