lab techniques

Question 1

PCR

Answer 1

-amplify a desired fragment of DNA

steps:

1. denaturation by heating
2. annealing- during cooling, excess premade DNA primed anneal to a specific sequence of each strand to be amplided
3. elongation- heat stable DNA pol, replicates the DNA sequence following each primer

Question 2

agarose gel electophoreis

Answer 2

used for size separation of PCR products against a DNA ladder
-smaller molecules travel farther

Question 3

SNoW DRoP

blotting techniques

Answer 3

southern - DNA
Northern - RNA
Western - Protein

Question 4

Southern blot

Answer 4

• A DNA sample is enzymatically cleaved into smaller pieces, elctrophoresed on a gel and then transferred to a filter.
• the filter is then soaked in denaturant and subsequently exposed to a radiolabeled DNA probe that recognizes and anneals to its complementary strand.
• the resulting double stranded, labeled piece of DNA is visualized when the filter is exposed to film

Question 5

northern blot

Answer 5

• similiar to southern blot but is for RNA

- useful for studing mRNA levels which are reflective of gene expression

Question 6

Western blot

Answer 6

sample protein is separated via gel electrophoresis and transferred to a filter

• labeled antibody is used to bind to relevant protein
• confirmatory test for HIV after a positive ELISA

Question 7

Southwestern blot

Answer 7

-identifies DNA binding proteins (Transcription factors) using oligonucleotide probes

Question 8

microarrays

Answer 8

• thousands of nucelic acid sequences are arranged in grids on glass or silicone
• DNA or RNA probes are hybridized to the chip and the scanner detects the relative amounts of complementary biding
• used to profile gene expression of thousands of genes simultaneously to study certain diseases and treatments
• able to detect single nucleotide polymorphisms and copy numver variations for:
• genotyping
• clinical genetic testing
• forensic anyalysis
• cancer mutations
• genetic linkage analysis

Question 9

ELISA enzymle linked immunosorbant assay

Answer 9

• detect presence of either a specific antigen (direct) use a test antibody
• detect presence of specific antibody (indirect) - use a test antigen

if target substance is presence - color change

Question 10

FISH flourescence in situ hybridization

Answer 10

-used for specific localization of genes and direct visualization of anomlies eg microdeletions at molecular level - when deletion is to small to be visualized by karyotype

Question 11

cloning

Answer 11

production of recombinant DNA
-isolate eukaryote mRNA (post-mrna processing)
-expose to reverse transcriptase to makde cDNA (lacks introns)
-insert cDNA into bacterial plasmids containign antibiotic resistance genees
-transform recombinant plasmid into bacteria
-surviving bacter on antibiotic medium producte cDNA
-

Question 12

knock out mouse

Answer 12

removing a gene, taking it out

Question 13

knock in mouse

Answer 13

-inserting a gene

• random insertion of gene into mouse genome
• targeted insertion of deletion of gene though homologous recombination with mouse gene

Question 14

RNA interference

Answer 14

• dsRNA introduced
• infect human cells
• when it separates it promotes degradation of target mRNA, knwocking down gene expression