Lecture 3: Tools of Molecular Pathology

Question 1

Pesudogenes

Answer 1

Non-functional gene-related sequence

Question 2

DNA can be selectively amplified by:

Answer 2

PCR (very quick - hours) and cloning (takes long)

Question 2

PCR process:

Answer 2

Cyclical process of heating and cooling to denature, anneal and enzymatically amplify DNA.

Step 1: Denaturation - heated (95˚C) allowing DNA strands to separate.
Step 2: Annealing - cooled (55-70˚C) to allow DNA primers to bind to specific interested regions
Step 3: Extension - heated again (70˚C), DNA polymerase binds and synthesizes new DNA strands.

Cycle is repeated 30 times to exponentially create a billion copies.

Question 2

Applications for PCR:

Answer 2

• Substitute for cloning (chopping up DNA and inserting it into a bacterial plasmid).
• Targeted/specific sequence amplified.
• Can selectively detect DNA sequences not normally present in tissue (i.e. viruses).
• Analysis of highly degraded DNA samples.

Question 3

Gel Electrophoresis

Answer 3

• Can use to separate molecules based on their size and conformation (smallest move further).
• Molecules move down to the positive side (since its negatively charged).
• Different gels are used for molecules with ranging nucleotide sizes.

Question 3

Gels in Gel Electrophoresis

Answer 3

Polyacrylamide gel: for single stranded DNA molecules less than 500 nucleotides

Agarose gel: more porous gels for molecules 300-20,000 nucleotides long.

Pulsed-field gel: for long DNA molecules.

Question 4

Application of Gel electrophoresis

Answer 4

• It can be used for genetic testing (i.e. if x or z is the mother of y).
• For DNA fingerprinting.
• Tells us if the parent got a disease genetically or if it was environmentally acquired.

Question 5

DNA Sequencing

Answer 5

• Allows you to read the nucleotides.

PCR containing fluorescent, chain-terminating dideoxynucleotide triphosphates.

Parent and offspring’s DNA samples are placed into a capillary electrophoresis.

Electrophoregram