7 Genes and Genomes Flashcards
(50 cards)
How do geneticists replicate - measure - store - change DNA ?
replicate = PCR
measure = Gel electrophoresis
store = vectore libraries
change = mutagenises/CRISPR
how do geneticists determine DNA sequence
Sangar or NExt gen sequencing
How do geneticists determine when and where DNA is used
qPCR and in situ hybridization
what are some facts about PCR
○ Done inside test tubes (invitro)
○ Amplifies DNA fragments
○ Requires primers
what are some uses of PCR
○ DNA fingerprinting
§ Forensics
§ Paternity testing
§ Prenatal screening
○ Mutation detection
○ Detect specific pieces of DNA
what are PCR requirements
○ Template DNA-from cells
○ 2 specific short Primers
§ Need forward and reverse primers
○ dNTPs
§ to make more DNA
○ Buffer
§ make environment ideal for enzymes
○ DNA polymerase
§ Synthesize DNA
§ Taq polymerase specifically (resistant to high temp)
what are the steps to PCR
○ denaturation
§ Heat to separate DNA strand (95 deg)
○ annealing
§ Cool so primers bind (60 deg)
○ extension
§ Heat up to taq polymerase ideal temp (72 deg)
○ Taq synthesizes DNA (step is short bc we don’t want to synthesize the whole strand)
what are intercalators in measuring DNA
§ Bind in DNA
§ They are fluorescent so we can see the DNA (it glows)
what are vectors
○ Small, transferrable, and replicable DNA molecs
what are 2 types of vectors
§ Plasmids (bacteria)
□ Circular DNA
□ Vary in size
§ Phage (virus)
□ Moonlander type shape
□ Use their genome to replicate our DNA we snuck into it
what are some traits of gene cloning
○ In vivo (in life) system
○ Insert into viable host molecule
§ This is our vector
○ Can be extracted and purified
what are traits of engineered plasmids
○ starts at Ori
§ Origin of replication
§ How plasmid replicates itself
§ Where polymerase binds
○ AmpR (ampicillin resistant)
§ Selective marker
§ Prevents bacteria from dying from ampicillin
○ Has polylinker site
§ Where we put our DNA insert
how do we fuse DNA into recombinant molecs
○Use Restriction enzymes
§ Type of endonuclease
§ Make site specific cuts in DNA
□ Cuts at the restriction site
□ Cuts double stranded DNA
how does Ligation of an insert into a vector work
- circular plasmid (vector) opened up with a restriction digestion
- insert placed into opened DNA with restriction enzymes to make matching end
- ligase + plasmid backbone inserted
how do we get our ligated DNA product (circular plasmid DNA we changed) in the cell
- heat shock or electroporation (electric shock) so the membrane will allow our vector in
what is the cloning rationale (steps)
1) Extract and isolate gene from cell
a. PCR
b. Gel
c. Restriction enzyme
2) Ligation
3) Clone into the vector model
a. Viral or plasmid
4) Transformation
5) Introduce vector into host cell
a. Bacteria replicates
6) extraction
a. Retrieve gene from host
TF What needs to be in both the vector and the insert
restriction enzymes
what is the pahge life cycle that we want in regards to cloning
The lytic cycle
what is the lytic cycle in a phage lifecycle
After out phage with out DNA is inserted into the cell
- our DNA will replicate and make many viral chromosomes
- our phages will be created in the cell
- at the end there is no circular DNA in the cell and our phages will burst from the cell wall
why do we not care about the lysogenic cycle
because we want them right away and the lysogenic cycle takes time
how do we find the bacteria that we want from the cells
Kill everything else
○ 2 steps to this process
§ Select for plasmid
□ Ensure that only the bacteria with the plasmid will survive (Antibiotic resistance)
§ Selection for insert
□ Identify bacteria with recombinant plasmids
□ Blue/white selection
why does the bacteria we want not die when we add the antibiotics
our bacteria has AmpR (ampicillin resistance)
what does the LacZ gene produce
Beta-galactosidase
TF LacZ turns X-Gal Red
F, turns it blue
