Molecular Diagnostic

Question 1

Blotting technique

Answer 1

1) gel electrophoresis to separate material
2) transferred to immobilized membrane
3) add target probe to fluorescent

Question 2

Pcr

Answer 2

1) denature dna
2) primer added in 3-5’ direction
3) anneal
4) add taq polymerase with dntps

Question 3

RFLP in sickle cell

Answer 3

Normal: 3 del restriction cuts
In sickle cell: only 2 restriction sites

1) restriction sites on del
2) gel electrophoresis and southern blotting
3) add fluorescent probes on beta globulin

Question 4

Which proteins do recombinant?

Answer 4

Insulin, erythropoietin, growth hormone, clotting factors, vaccines

Question 5

For recombinant, what type of dna do you use?

Answer 5

cDNA

Question 6

How do you improve insulin through recombinant DNA?

Answer 6

Normal insulin has proline on 28 and lysine on 29 in C terminus of B chain

Lispro switches these two AAs

Insulin aspart replaces Proline 28 with aspartic acid

Lispro and insulin are faster acting

Question 7

Western blotting

Answer 7

Uses SDS-page

• Gel electrophoresis but measuring the size of protein
• transfer to nitro cellulose membrane
• Bind a primary anti-body
• Add secondary anti-body that has enzyme tag
• Add substrate to react with enzyme to change color