Biochemical/Genetic Lab Methods

Question 1

What does a PCR solution contain?

Answer 1

DNA primers specific for DNA region, DNA sample of interest, heat stable DNA polymerase, deoxyribonucleotides

Question 2

Steps of PCR?

Answer 2

Denaturation, Annealing, Replication, Repeat

Use gel electrophoresis to separate components

Question 3

Clinical uses of PCR?

Answer 3

1. high specificity proving bacterial/viral infection
   - confirmatory test for HIV infection (after ELISA) –> know amount of viral DNA integrated into host DNA
   - positive earlier in disease course; doesn’t require competent immune system
   - always use for newborn of HIV+ mother/when early detection is required
2. genetic ID - paternity testing with variable number or short tandem repeats
3. finding direct mutations

Question 4

Process of RT-PCR?

Answer 4

• measure amount of RNA in sample

- solution has reverse transcriptase, RNA, dNTPs, primers for specific sequence, heat stable DNA polymerase

Question 5

Clinical uses of RT-PCR?

Answer 5

-HIV viral load –> transcriptional activity of virus by knowing amount of RNA present

Question 6

Clinical use of Southern blotting or Northern blotting?

Answer 6

Southern blotting - visualize restriction length polymorphisms (RFLPs) for genetic testing
Ex: sickle cell anemia mutation results in loss of endonuclease restriction site

Northern blotting - measure if particular DNA region is being expressed –> test for fragile X, increased expression of FMR1 gene, trinucleotide repeat disorders

Question 7

Clinical use of microarrays?

Answer 7

• analyze cancer cell gene expression by probing for thousands of different mRNAs simultaneously
• can be used to guide therapy, determine probability of tumor becoming malignant compared to the general population

Question 8

Clinical use of cloning to make recombinant proteins?

Answer 8

-make recombinant insulin, factor VIII, bacterial factors for vaccination