DNA and RNA analysis

Question 1

Molecular hybridization

Answer 1

• use to know the location of
• Fluorophore (donor), Quencher (acceptor)are attached to ends of DNA
• adding hot temperature with probe = denaturation
• when unfolded F is far enough away from Q that it can emit a fluorescent colour

Question 2

Formaldahyde

Answer 2

• fixes cell in place so when you put the probe in and heat the DNA it will separate
• one you start to cool DNA the probe will go in and is complementary to a strand
• when probe hydrolyzes it linearizes (more stable = more bonds)

Question 3

Gel electrophoresis

Answer 3

• takes advantage of charge on DNA
• DNA samples are loaded into wells, power is turned on and DNA fragments migrate through gel
• fragments are separated by size - largest at negative, smallest at Positive

Question 4

Restriction enzymes

Answer 4

• group of endonucleases produced in bacteria as a means of destroying foreign DNA
• function by cleaving DNA at specific double-stranded sequences known as restriction sites

Question 5

Where do restriction sites occur

Answer 5

• randomly, at many distinct locations throughout the genomes
• REs can target and cleave DNA at the particular locations where a restriction site occurs

Question 6

EcoRI

Answer 6

restriction enzyme that recognizes GAATTC sequence (restriction site)
- endonuclease chops DNA to produce sticky ends

Question 7

SmaI

Answer 7

restriction enzyme that recognizes CCCGGG (restriction site)
- endonuclease that cuts right down the middle and makes blunt ends

Question 8

Multiple cloning site

Answer 8

region with many restriction sites into which exogenous or external DNA is inserted

Question 9

origin of replication

Answer 9

enables exogenous or external DNA is inserted

Question 10

selectable marker

Answer 10

enables researchers to select cells that contain a plasmid

Question 11

cloning plasmid

Answer 11

• many plasmids naturally occurring in bacteria and yeast have been edited and engineered to be optimized for cloning use
  3 components…
• multiple cloning site (MCS)
• origin of replication (ORI)
• selectable marker (ex. ampr)

Question 12

How to insert a gene of interest in plasmid

Answer 12

1) Digest
2) Ligation
3) Transformation
4) Selection

Question 13

1) Digest

Answer 13

plasmid and foreign DNA are cleaved with restriction enzymes forming complimentary sticky ends

Question 14

2) Ligation

Answer 14

DNA fragments and plasmids hybridize at sticky ends. DNA ligase forms phosphodiester bonds to “seal nicks” in each strand

Question 15

3) Transformation

Answer 15

the ligated plasmid is mixed with bacterial cells under conditions to optimize transformation

Question 16

4) selection

Answer 16

only cells containing the plasmid will grow on ampicillin plates forming colonies which all have plasmid
- transformed cell survives, cells that don’t take up plasmid die on plates

Question 17

Polymerase chain reaction

Answer 17

• technique for rapidly producing many copies of a specific segment of DNA
  Step 1: denaturation - 95C
  Step 2: Primer annealing - 52C
  Step 3: Elongation - 72C

Question 18

PCR - denaturation

Answer 18

double-stranded DNA is separated into single strands

Question 19

PCR - primer annealing

Answer 19

specific primers are added to their complementary strand - primers are usually 20 base pairs long
Forward primer: anneals to the bottom strand and has the same sequence as the top strand
Reverse Primer: anneals to the top strand and has the same sequence as the bottom strand

Question 20

PCR - Elongation

Answer 20

DNA polymerase copies template DNA
- TAQ polymerase used in PCR comes from thermal bacteria that lives at high temp, so their proteins will not denature at high temp

Question 21

Next generation sequencing

Answer 21

genomic DNA to be sequenced is first fragmented, then adaptor sequences are added to each fragment which are then fixed to a solid surface.
- PCR is used to amplify each fragment so that each bead contains thousands of copies

Question 22

Addaptor

Answer 22

short DNA sequence you know the sequence of