Lec 12: Lab Techniques

Question 1

Hybridization

Answer 1

1. Utilizes complementary base pairing / hydrogen bonding between antiparallel nucleotides (DNA and RNA, DNA and DNA, RNA and RNA)
2. Tm: melting temperature (when dsDNA becomes ssDNA)

Question 2

PCR

Answer 2

1. Heating to 90 degrees C to get ssDNA
2. Can re-anneal the strands together by dropping the temperature (to about 50 degrees C):

Question 3

Primers

Answer 3

1. Used by DNA polymerase in DNA replication:
   1a. DNA replication: RNA primary is made by primase and removed later
2. Lab uses DNA primers: are short sequences (18-25nt), specific, ssDNA, complementary to target

Question 4

Probe

Answer 4

1. DNA or RNA, used to bind and identify specific sequences in mixture (to find presence and location)
2. These are longer (300-700nt) and complementary to specific target sequence
3. The way to label probes is through radioactivity (using 32P in PO4-), colour metric (enzyme reaction creates colour), fluorescence (glow)

Question 5

The way to make antibodies

Answer 5

1. Make it using an animal:
   1a. Inject your antigen (protein) into the animal, make the antibody (primary), take antibody and put into animal and make anti-antibody (secondary) which causes signal amplification

Question 6

Epigenetics

Answer 6

1. DNA packing, changes gene expression
2. DNA methylation: gene silencing

Question 7

DNA techniques: gel electrophoresis

Answer 7

1. Use agarose gel
2. Separate DNA fragments by size and molecular mass with a nonspontaneous electrolytic cell

Question 8

DNA techniques: restriction enzymes

Answer 8

1. Restriction enzymes are endonucleases that cut DNA (palindromes) within strand

Question 9

DNA techniques: PCR

Answer 9

1. Photocopy any dsDNA

Question 10

DNA techniques: Cloning

Answer 10

1. Copy DNA (PCR), cut specific ends with restriction enzymes and purify in gel
2. DNA ligase into plasmid/vectors and add into new organism

Question 11

DNA techniques: southern blot

Answer 11

1. Detect specific DNA

Question 12

DNA techniques: RFLP

Answer 12

1. Detect mutations in DNA

Question 13

DNA techniques: DNA sequencing

Answer 13

1. Track nucleotides added

Question 14

DNA techniques: FISH

Answer 14

1. Fluorescence in situ

Question 15

RNA techniques: gel electrophoresis

Answer 15

1. Acrylamide gel (RNA is smaller)

Question 16

RNA techniques: RT-PCR or q-PCR(reverse transcriptase)

Answer 16

1. Make dsDNA from ssRNA, do PCR and compare gene expression by quantifying with q-PCR

Question 17

RNA techniques: northern blot

Answer 17

1. Compare gene expression between samples

Question 18

RNA techniques: RNA ISH

Answer 18

1. RNA in situ hybridization

Question 19

RNA techniques: microarray

Answer 19

1. Computer analysis

Question 20

Protein techniques: gel electrophoresis

Answer 20

1. Acrylamide (Smaller)
2. Types
   2a. Isoelectric focusing
   2b. Native gel
   2c. SDS page
   2d. Reducing gel

Question 21

Protein techniques: western blot

Answer 21

1. Separate proteins and detect w antibodies

Question 22

Protein techniques: chromatography

Answer 22

1. Separate from a mixture a group/single protein

Question 23

Protein techniques: immunocystochemistry

Answer 23

1. Detect proteins /epitopes w antibodies