Lect 27 - PCR - how it works and how it's used in diagnosis

Flashcards about PCR, its mechanisms, and its applications in various diagnostic procedures.

What is DNA polymerase?

Enzyme used by cells to replicate DNA, adding complementary nucleotides in the 5' to 3' direction to the 3' end of each primer.

What is the chain reaction in PCR?

By using two short primers on either side of the DNA in interest, the region can be selectively copied through repeated melting and cooling.

What is the most common cause of cystic fibrosis?

Deletion of 3 base pairs that code for phenylalanine amino acid (deltaF508 allele, 70% of cases).

How does gel electrophoresis show heterozygosity vs. homozygosity after PCR?

Individuals who are heterozygous for an allele present 2 bands, while homozygous individuals present 1 band.

Why is preimplantation genetic diagnosis possible without harming the foetus?

Removing one cell from the embryo for PCR will not affect the foetus because the cells have not yet specialised.

How is PCR used in preimplantation genetic diagnosis of IVF embryos?

To amplify the relevant region of DNA from each embryo cell biopsy and check for the presence of each parent’s variant to select healthy embryos for implantation.

Give examples of diseases/aneuploidies that are generally considered when there is a known risk of fatal or severely debilitating monogenic genetic disease or aneuploidy where IVF may be used.

Cystic fibrosis, sickle cell disease, duchenne muscular dystrophy, huntington’s disease, trisomy 13 (Patau syndrome), trisomy 18 (Edwards syndrome), trisomy 21 (down syndrome).

Name two invasive procedures that test for genetic disorders but have a risk of miscarriage.

Amniocentesis and chorionic villus sampling.

What is cell-free DNA (cfDNA)?

Fragmented DNA freely circulating in the bloodstream, some of which comes from placental cells in pregnant women.

What discovery did Dr. Lo make regarding cell-free DNA?

Fragments of an unborn baby’s DNA can be amplified from the mother’s plasma.

What was initially detected in the plasma of pregnant women carrying a male foetus?

Fragments belonging to the Y chromosome.

Where does fetal DNA come from in maternal plasma?

The DNA comes from dying placental cells and is a normal process where the DNA makes up to 10% of cell free DNA in maternal plasma.

Because fetal DNA is likely shorter what can be done to amplify more effectively?

By selecting for shorter fragments (<150bp) of foetal DNA.

What is necessary in cfDNA for non-invasive prenatal diagnosis (NIPD) when we don’t know the sequence?

Ligate ‘linkers’ on the ends of the DNA fragments enabling primers to be designed to these known sequences.

From whom is mitochondrial DNA inherited?

Mitochondrial DNA is inherited from the mother.

What is the difference between nuclear and mitochondrial DNA?

Nuclear DNA is within the nucleus containing all genetic instructions for development. Mitochondrial DNA is outside the nucleus and inherited only from the mother.

From whom the DNA comes in ‘three parent babies’?

Nuclear DNA from the parents and mitochondrial DNA from an egg donor.