Molecular Genetics

The Molecular Genetics department conducts studies aimed at identifying mutations and/or genotypes that cause a hereditary disease for clinical purposes. In addition to detecting disease-causing mutations, carrier analysis for a genetic disorder and susceptibility analysis for multifactorial disorders are also carried out. Single-gene disorders (Thalassemia, FMF), Multifactorial Disorders (Cancer), and Mitochondrial Disorders (LHON) are studied in the Molecular Genetics department.

Some of the techniques used in our center for routine genetic analyses are listed below.

• RFLP (Restriction Fragment Length Polymorphisms)
• PCR (Polymerase Chain Reaction)
• Sequencing (Sequence Analysis)
• RT-PCR (Real Time PCR)
• Heteroduplex Analysis

RFLP

This method is based on cutting DNA with restriction endonucleases (enzymes that cleave DNA at specific sites) and analyzing the resulting fragments of different sizes. It is widely used in genotyping and polymorphism studies.

PCR

PCR is the enzymatic amplification of a specific DNA region using short initiator sequences called primers. It enables millions of copies of a specific DNA fragment to be generated.

• Denaturation: In the first step, the double-stranded structure of the DNA molecule is separated using high heat. This is typically applied at 94-97°C for 15-60 seconds.
• Annealing: Following denaturation, oligonucleotide primers bind at lower temperatures to their complementary regions on the separated single-stranded DNA. This typically occurs at 47-60°C for 30-60 seconds; in regions with a high G/C content, the annealing temperature can rise to as high as 68°C.
• Elongation: In the final step, the temperature is raised to 72°C, allowing the DNA polymerase enzyme to extend the complementary DNA strand. The duration can range from approximately 30 seconds to 3 minutes, depending on the polymerase used and the length of the DNA fragment being amplified.

Sequence Analysis

Sequence Analysis is the process of determining the order of nucleotides in a DNA region whose sequence is unknown. Certain nucleotide changes occurring in genes are called mutations and cause various hereditary diseases. The gene region responsible for a disease is screened using Sequence Analysis to identify the exact location of the mutation within the gene. Advances in sequencing devices have shortened processing times. Sequencing methods are used for definitive diagnostic evaluation in many single-gene disorders, such as Beta Thalassemia and FMF.

Real Time-PCR

Real-time PCR is a system that monitors the reaction step by step, based on the fluorescent light emitted once a sufficient quantity of product accumulates during each PCR cycle. It can measure the quantity of PCR products simultaneously. It is widely used in infection testing and hematology.

Heteroduplex Analysis

Heteroduplex analysis is a method that identifies sequence differences between reference DNA (normal DNA) and DNA containing a mutation. The reference and test DNA are mixed, denatured using heat, and then cooled to allow the DNA strands to hybridize. If there is a difference between the two DNA sequences, the two bases will fail to pair at that point, forming a heteroduplex. Because heteroduplexes migrate more slowly than homoduplexes during electrophoresis, they can be easily distinguished.