PCR, Restriction Analysis and Electrophoresis Lab

Flashcards about PCR, Restriction Analysis, and Electrophoresis Lab, Sickle Cell Disease Diagnosis

What is the function of hemoglobin?

Hemoglobin carries oxygen through the body.

What is the composition of hemoglobin?

Hemoglobin is made of four polypeptide chains: 2 alpha and 2 beta.

What causes sickle cell disease?

A mutation in the beta chain of hemoglobin causes the soluble Hb molecules to stick together and form fibers.

What specific change occurs in the sickle hemoglobin mutation?

A single nucleotide change in the HbA gene converts a Glutamic acid to a Valine in the beta polypeptide chain.

What is the impact of the amino acid change in sickle cell disease?

The mutation changes a hydrophilic amino acid to a hydrophobic amino acid, creating a hydrophobic patch on the surface of Hb.

What is the result of the hydrophobic patch on hemoglobin in sickle cell disease?

HbS molecules aggregate and red blood cells sickle.

What specific nucleotide change causes sickle cell disease?

The mutation that causes sickle cell is a change from an adenine (A) to a thymine (T) in position 20 of the beta-globin coding sequence.

How can we test for the sickle cell mutation?

We can test for this mutation using a restriction enzyme.

What is the function of restriction enzymes?

Restriction enzymes recognize a very specific sequence of DNA, and when the sequence is recognized, the enzyme cuts the DNA in two.

What sequence does the restriction enzyme DdeI cut?

The restriction enzyme DdeI cuts the sequence CTNAG.

In what sequence is CTNAG present?

The sequence CTNAG is only present in the HbA, or normal, beta-globin sequence, so only that sequence will be cut by the restriction enzyme.

What is the goal of today's lab?

To use PCR, Restriction analysis with DdeI and Electrophoresis, to test whether each member of the Ryan family has sickle cell disease, the sickle cell trait, or is unaffected by sickle cell.

What are the steps of PCR?

Denaturation (95C), Annealing (50-60C), Extension (72C).

What is the usage of restriction enzymes in bacteria?

They use them to destroy foreign DNA such as what comes in to the cell when a bacteriophage infects it.

Why are restriction enzymes molecular scissors?

They can be used to create recombinant DNA, by cutting and pasting DNA from different sources.

What are restriction sites?

Restriction sites are palindromes: when the top strand is read in one direction it has the same sequence as the bottom strand read in the opposite direction.

What is gel electrophoresis?

Gel electrophoresis separates molecules based on size.

Why is agarose gel used for separating DNA?

Agarose is a polysaccharide extracted from seaweed, that forms a porous matrix that molecules are pulled through, that allow to separate the DNA by size

How does molecules separate in agarose gel?

Larger molecules have more trouble making it through the gel pores and move slower, while smaller molecules have an easier time making it through the gel pores and move faster.