Molecular pathology

Molecular pathology

Lecture 4

Definition The study of biochemical and biophysical cellular mechanisms as the basic factors in disease.

anatomic pathology, clinical pathology, molecular biology, biochemistry, proteomics and genetics

Use of nucleic acid based tests to determine diagnosis or prognosis

Includes hybridization,ISH, blotting and sequencingGenerally doesn‟t include protein assays or antibodydetection (however some define it more broadly).The field typically includes both molecules testing intubes and slides (cytogenetics).

• anatomic pathology, clinical pathology, molecular biology, biochemistry, proteomics and genetics

• anatomic pathology, clinical pathology, molecular biology, biochemistry, proteomics and genetics

Uses • Diagnosis• Prognosis • Prenatal testing• Pharmacotherapy

– Pharmacogenetics – Pharmacogenomics

Watson and crick (1953)

• They described DNA model as long double helix shaped with its sugar phosphate backbones on the outside and its bases on the inside.

• The two strands run in opposite direction and are anti-parallel to each other.

• Stabilized by hydrogen bonds between bases.• They were awarded nobel prize in 1962.

Techniques: PCR PCR was first conceived in 1983 by Kary Mullis, a molecular biologist who received a Nobel Prize for the discovery 10 years later

A PCR (Polymerase Chain Reaction) is performed in order to make a large number of copies of a gene. Otherwise, the quantity of DNA is insufficient and cannot be used for other methods such as sequencing.

A PCR is performed on an automated cycler, which heats and cools the tubes with the reaction mixture in a very short time.

Performed for 30-40 cycles, in three major steps: 1)denaturation, 2)annealing, and 3)extension.

Techniques: PCR1) Denaturation at 94°C :

During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions halt.

2) Annealing at 54°C : The primers are freely moving due to Brownian motion. Ionic bonds are

constantly formed and broken between the single stranded primer and the single stranded template.

Primers that fit exactly will have stable bonds that last longer.

The polymerase attaches onto a piece of double stranded DNA (which is template and primer), and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.

Techniques: PCR• 3) Extension at 72°C :

• This temperature is ideal for the polymerase. The primers, which have a few

bases built in, have a stronger ionic attraction to the template than the forces

breaking these attractions.

• Primers that are on positions with no exact match, loosen their bonds again

(because of the higher temperature) and do not extend the fragment.

The bases (complementary to the template) are coupled to the primer on the

3' side (the polymerase adds dNTP's from 5' to 3', reading the template from

3' to 5' side, bases are added complementary to the template)

Techniques: PCR• At the end of a PCR, the product must be checked before it is used in

further applications. This is to confirm:

• There is a product formed: Not every PCR is successful. The quality of

the DNA is poor, that one of the primers doesn't fit.

• The product is of the right size: It is possible that there is a product, for

example a band of 500 bases, but the expected gene should be 1800 bases

long. In that case, one of the primers probably fits on a part of the gene

closer to the other primer. It is also possible that both primers fit on a

totally different gene.

• Only one band is formed: It is possible that the primers fit on the desired

locations, and also on other locations. different bands in one lane on a gel.

Applications of PCR• 1) Diagnosis of Disease: Linkage analysis,

detection of mutant alleles, diagnosing infectious agents, epidemiological studies

• 2) Forensics: paternity testing, DNA typing for identification, criminal investigations.

• 3)Recombinat DNA engineering• 4) DNA sequence determination• 5) new gene isolation• 6) Anthropological studies: population genetics,

migration studies.• 7) Evolution studies• If you need to look at 100 genes is PCR a good

approach?

Applications of PCR• 1) Diagnosis of Disease: Linkage analysis, detection

of mutant alleles, diagnosing infectious agents,

epidemiological studies

• 2) Forensics: paternity testing, DNA typing for

identification, criminal investigations.

• 3)Recombinat DNA engineering

• 4) DNA sequence determination

• 5) new gene isolation• 6) Anthropological studies:

population genetics, migration studies.

• 7) Evolution studies