MEDICAL BIOLOGY, HUMAN ANATOMY, PHYSIOLOGY AND PATHOLOGY - Ya.I. Fedonyuk 2010

BIOLOGY

SECTION 1. BIOLOGICAL BASES OF HUMAN VITAL ACTIVITY

1.4. ONTOGENETIC LEVEL OF LIFE ORGANIZATION

1.4.2. Fundamentals of human genetics

Molecular-genetic method (DNA analysis)

Stages of the method: 1. Obtaining DNA (or RNA) samples, which is the initial starting step for all Methods. There are two possible options for this stage:

a) isolation of total or genomic DNA from Cells;

b) Amplification of specific fragments that can be analyzed using the Polymerase Chain Reaction.

Any nucleated cells can serve as a source of genomic DNA. In practice, peripheral Blood Leukocytes, chorion, amniotic cells, and fibroblast cultures are most commonly used. Sometimes, diagnosing a disease or a heterozygous state requires only a small DNA fragment, which must be amplified. This can be achieved using the polymerase chain reaction.

Polymerase chain reaction (PCR) is a method for in vitro DNA amplification using DNA polymerase, which synthesizes mutually complementary DNA strands. A prerequisite for PCR is knowing The nucleotide sequence of the amplified DNA fragment, to the ends of which complementary oligonucleotide primers (initiators) attach. The synthesis of polynucleotide strands on the studied DNA fragment occurs within the region between the two primers. Thus, millions of DNA copies can be obtained.

DNA Restriction into fragments is performed by restriction Enzymes (Restriction Endonucleases). They cleave double-stranded DNA at strictly defined nucleotide sequences 4-6 Base Pairs long, which are characteristic of each fragment.

Electrophoresis of DNA fragments ensures their Separation when distributed On the surface of an agarose or polyacrylamide gel. Upon completion of electrophoresis, each DNA fragment occupies a specific position as a discrete band at a particular Location in the gel. The fragment length is compared with a standard DNA ladder of known sizes.

Visualization and identification of DNA fragments in the gel can be The final stage of Diagnostics or a necessary element of subsequent analysis. Visualization of DNA fragments is achieved through various staining methods. Identification of specific fragments is a more complex task, which is resolved using Southern blot Hybridization. The Procedure consists of the following stages:

1) DNA Denaturation to obtain single-stranded fragments;

2) transfer of DNA from the gel to a nitrocellulose or nylon filter;

3) immobilization (fixation) of DNA strands on the filter;

4) hybridization of these fragments with a specific oligonucleotide synthetic probe (or a cloned DNA fragment) whose nucleotide sequence is complementary and labeled with a radionuclide or fluorescent tag;

5) exposure of the filter to X-ray film to reveal radioactively labeled regions; non-radioactive identification is performed using luminescence Cell/15.html">Microscopy based on the fluorescence of the stained area or indirectly using Antibodies.

of the stained area or indirectly using antibodies.