Lab Techniques

Question 1

PCR (polymerase chain reaction)

Answer 1

• Amplifies fragment of DNA; do repeatedly to amplify

Steps:

1) Denature DNA (heat to make 2 strands)
2) Annealing - while cooling, put in premade DNA primers to anneal to specific sequence on each strand
3) Elongation - DNA polymerase replicates DNA sequence of each primer

Question 2

Agarose gel electrophoresis

Answer 2

Used to separate separation of PCR products

• Small travel further
• Compare to DNA ladder

Question 3

Blotting procedures

Answer 3

SNoW DRoP:

• Southern = DNA
• Northern = RNA
• Western = protein

Question 4

Southern blot

Answer 4

DNA cleaved to small pieces w/ enzymes

• Electrophoresed on gel –> transfer to filter
• Filter soak in denaturant - exposed to radiolabeled DNA probe (anneals complement strand)
• Expose to film, dsDNA labeled seen

Question 5

Northern blot

Answer 5

Similar to southern, but done for RNA

- used for studying mRNA levels (reflects gene expression)

Question 6

Western blot

Answer 6

Sample protein separated w/ gel electrophoresis –> to filter

• Labeled antibody binds specific protein
• Use to confirm HIV after +ELISA

Question 7

Southwestern blot

Answer 7

ID DNA-binding proteins

- Use these to label oligoNT probes

Question 8

Microarrays

Answer 8

Used to profile gene expression levels in thousands of genes at once to study diseases/tx
- Detect SNPs, CNVs; good for genotyping, clinical genetics tests, forensics, cancer mutations, genetic linkage analyses

Question 9

ELISA (enzyme-linked immunosorbent assay)

Answer 9

Detect specific antigen (direct) or antibody (indirect) in blood

• Indirect ELISA - uses antigen test to see if antibody present
• Direct ELISA - uses antibody to see if antigen present
• Both use secondary antibody w/ color-generating enzyme, so + result = color reaction
• Often first used for HIV testing

Question 10

FISH (fluorescence in situ hybridization)

Answer 10

Fluorescent DNA/RNA probe binds specific gene site on chromosome

• Used to localize genes/see anomalies (microdeletions) at molecular level
• Fluorescence = gene present
• No fluorescence = gene deleted

Question 11

Cloning mthods

Answer 11

Make recombinant DNA that’s self-perpetuating
Steps:
1) Isolate mRNA you want
2) Expose mRNA to reverse transcriptase –> cDNA (no introns)
3) Insert cDNA into bacterial plasmids –> antibiotic resistance genes
4) Transform recombinant plasmid into bacteria
5) Survive bacteria on antibiotic medium make cDNA

Question 12

Gene expression modifications

Answer 12

Transgenic mice:

• Random insertion of gene into mouse
• Targeted insertion or deleted gene w/ homologous recombination w/ mouse gene

Question 13

Cre-lox system

Answer 13

Manipulate genes at developmental points in embryology

Question 14

RNA interference

Answer 14

dsRNA made complementary to mRNA sequence of interest

• Put into humans -> dsRNA separates and promotes degradation of targeted mRNA
• Knocks down gene expression

Question 15

Karyotyping

Answer 15

Metaphase chromosomes stained, ordered, #ed

- Used to dx chromosomal abnormalities (trisomies, sex chromosome d/o)