Molecular Diagnostics

Question 1

Hybridization

Answer 1

ssDNA binds to another sequence that is either DNA/RNA. Used to detect the quantification of target DNA or RNA.

Question 2

Southern blotting

Answer 2

Probe and target are DNA.

Question 3

Northern blotting

Answer 3

Probe is ssDNA and target is mRNA.

Question 4

Western blotting

Answer 4

Target is protein. Used to measure amount of protein/antibody.

Question 5

Eastern blotting

Answer 5

Target is PTMs. Detects PTMs on proteins.

Question 6

PCR overview (5)

Answer 6

1. Denature DNA.
2. Add primers to complimentary sequences in 3’-5’ direction.
3. Allow DNA to to reanneal.
4. Add dNTPs.
5. Add Taq polymerase to amplify.

Question 7

qPCR

Answer 7

Used to detect levels of infectious agents and levels of gene expression. Probe is usually a complimentary oligo with a fluorescent tag.

Question 8

RFLPs

Answer 8

Because individual genomes are very similar, but many bp differences occur in recognition sites for restriction enzymes. RFLPs are used in gel. Used often in forensic analysis, paternity testing.

Question 9

VNTRs (variable number of tandem repeats)

Answer 9

Pattern of short tandem repeats varies in individuals. Used to identify and assess severity of inherited diseases.

Question 10

Normal human insulin AA positioning

Answer 10

Pro at 28 and Lys at position 29 at C terminus of B chain.

Question 11

Lispro changes to AA positioning

Answer 11

Reverse of normal positioning (Lys 28 and Pro at 29 at C terminus of B chain).

Question 12

Insulin aspart changes to AA positioning

Answer 12

Asp at 28 (instead of Pro).

Question 13

Benefits of Lispro and Insulin aspart

Answer 13

More readily absorbed and provides longer range of glycemic control when given with normal insulin.

Question 14

ELISA

Answer 14

Detects levels of specific antigens or AB concentrations.

Question 15

ELISA overview (5)

Answer 15

1. Microplate coated with a capture AB and immobilized to plate’s surface.
2. Protein target bound to AB when added to well.
3. Unbound molecules are washed away.
4. A second AB conjugated w/ HRP is added and binds to capture analyte.
5. Wash away HRP and then TMB is added to wells. Blue color is developed proportionally to the amount of analyte in sample.

Question 16

Indirect ELISA

Answer 16

Measures amount of AB in a sample.

Question 17

Sandwich ELISA

Answer 17

Measures amount of antigen in a sample.

Question 18

Immunoblotting (western blotting)

Answer 18

Uses SDS-PAGE to separate the proteins on a gel via electrical field

1. Transfer proteins from a gel to a nitrocelluose membrane to make surface exposed.
2. Add primary AB.
3. Add secondary AB. w/ enzyme tag.
4. Add substrate.
5. Color visualized.