DNA and RNA analysis Flashcards
Molecular hybridization
- 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
Formaldahyde
- 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)
Gel electrophoresis
- 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
Restriction enzymes
- 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
Where do restriction sites occur
- randomly, at many distinct locations throughout the genomes
- REs can target and cleave DNA at the particular locations where a restriction site occurs
EcoRI
restriction enzyme that recognizes GAATTC sequence (restriction site)
- endonuclease chops DNA to produce sticky ends
SmaI
restriction enzyme that recognizes CCCGGG (restriction site)
- endonuclease that cuts right down the middle and makes blunt ends
Multiple cloning site
region with many restriction sites into which exogenous or external DNA is inserted
origin of replication
enables exogenous or external DNA is inserted
selectable marker
enables researchers to select cells that contain a plasmid
cloning plasmid
- 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)
How to insert a gene of interest in plasmid
1) Digest
2) Ligation
3) Transformation
4) Selection
1) Digest
plasmid and foreign DNA are cleaved with restriction enzymes forming complimentary sticky ends
2) Ligation
DNA fragments and plasmids hybridize at sticky ends. DNA ligase forms phosphodiester bonds to “seal nicks” in each strand
3) Transformation
the ligated plasmid is mixed with bacterial cells under conditions to optimize transformation
4) selection
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
Polymerase chain reaction
- 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
PCR - denaturation
double-stranded DNA is separated into single strands
PCR - primer annealing
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
PCR - Elongation
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
Next generation sequencing
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
Addaptor
short DNA sequence you know the sequence of
