Diagnostic Techniques in Molecular Biology

- Polymerase Chain Reaction (PCR)

- Polymerase Chain Reaction (PCR) It is a rapid and sensitive method of amplifying

a target sequence of DNA. The method can be used to amplify DNA sequences from any source: bacterial, viral, plant or animal.

Required Components of PCR DNA template DNA

pool of free deoxynucleotides (dNTPs) DNA polymerase Primers thermocycler

PCR

Application: 1- To detect infection agents (ex. Latent viruses as HIV) 2- For genetic prenatal diagnosis. 3- To establish precise tissue types for transplantation. 4- To study evolution. 5- Forensic analysis of DNA samples. DNA isolated from a single hair is sufficient to determine whether the sample comes from a specific individual.

- Gel Electrophoresis

- Gel Electrophoresis
In electric current, The DNA fragment will

migrate according to its size toward the positive electrode because DNA contains negatively charged phosphate groups.

So, DNA segments can be separated. Agarose or polyacrylamide gel can be used.

Gel electrophoresis

 Restriction fragment length polymorphism (RFLP)

Principle:
Isolation of DNA. DNA is amplified by PCR. Amplified DNA is incubated with restriction endonucleases. Then electrophoresis. Visualization of different bands.

V- Hybridization and blot techniques

Definitions:
Hybridization:
It means specific reassociation between complementary strands of nucleic acids ( DNA&DNA, DNA&RNA, or RNA&RNA ).

Probe:
- It is a single stranded piece of DNA, - labelled (either with radio-isotope or with non-radioactive label), - its nucleotide sequence of is complementary to the target DNA.

Blotting techniques nucleic Blotting analysis can separate and detect specific
acid sequences or proteins in complex mixtures. Types:
1.

Southern blotting: detects DNA with labeled DNA probes. Northern blotting : detects RNA with labeled DNA probes. It is useful in determining whether a specific mRNA is expressed in a particular tissue. Western blotting: detects proteins by using labeled antibodies.

2.

3.

Principle:
1.

Extraction of DNA. Amplification of DNA by PCR. DNA is cleaved with a restriction enzyme. Agarose gel electrophoresis. Denature the DNA into single strands (by alkali). Transfer to nitrocellulose membrane (blotted). Hybridize with labeled probe. Band visualization. The labeled probes detect specific DNA sequences.

2.

3.

4.

5.

6.

7.

8.

Southern Blotting

Amplification of DNA:
-

Recombinant DNA. PCR.

Analysis of DNA:
- Electrophoresis only.
-

R.E. & Electrophoresis . (RFLP). R.E. & Electrophoresis & blot & probe(Southern blot).

Sickle Cell Disease

Sickle cell anemia is a disease of red blood cells. It is caused by a mutation in the hemoglobin gene. A single base change results in a single amino acid substitution. Sickle cell anemia is considered a recessive trait, since both chromosomes have to carry the mutation in order for the full blown disease symptoms to appear.

The sickle cell mutation eliminates a restriction enzyme site - the recognition site for the restriction enzyme MstII. To detect the sickle cell mutation, a patients DNA is digested with MstII and a Southern blot is performed using a probe corresponding to this region of the hemoglobin gene. The presence or absence of the sickle cell mutation can be determined based on the size of the fragment identified by the probe.

Cystic Fibrosis
The cause of the disease appears to be a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein involved in transporting ions across epithelial surfaces, such as the linings of the lungs and intestines.

cystic fibrosis is caused by deletion of 3 DNA bases missing codon in mRNA amino acid phenylalenine therefore missing from transmembrane protein in lungs. This mutation is detected by sequence analysis of PCR-amplified DNA, or by hybridization with mutation-specific probes.

Genetic testing of CFTR using PCR

PCR primer 1 Normal CFTR gene

PCR primer Mutant CFTR gene

Cystic fibrosis
1 1+ 2 2
1 is homozygous normal. 2 is homozygous mutated. 1+2 is carrier (heterozygou s mutated).

63 bp 60 bp