Molecular Cloning Flashcards
( ! ) Why is Molecular Cloning Important?
Hint: PODPEER
P - Purify & Amplify Frags
O - Obtain sequences
D - Determine Gene Struc & Reg
P - Perform Site-Directed MGenesis
E - Express & Purify protein; BIOCHEM and STRUCTURAL analysis
E - Enable genome analysis with OVERLAPPING CLONES
R - Reintroduce (transgenesis)
What do Type II REs do?
Cleave DNA at specific bases
What do Type II Methylases do?
Methylate DNA at specific bases
What do DNA Polymerases do?
Copy DNA from a PRIMER 3’ end
What do RNA Polymerases do?
Make an RNA copy from DNA from a promoter
Opp. of Reverse Transcriptase
What do Reverse Transcriptases do?
Make a DNA copy of RNA from PRIMER 3’ end
Opp. of RNA Polymerase
What do DNA Ligases do?
Join two DNA molecules//fragments
What do Exonucleases do?
Remove NTs from ends of DNA
Sort of Opp. of Terminal Transferases
What do Terminal Transferases do?
Add a homopolymer tail to the ends of DNA
Sort of Opp. of Exonucleases
What do Polynucleotide Kinases do?
Add a PHOSPHATE to the 3’ ends of DNA
Opp. of alkaline phosphatase
What do Alkaline Phosphatases do?
Remove terminal phosphates from DNA ends
Opp. of Polynucleotide Kinases
What do Type II REs recognise, and what do they Require?
Recognise SHORT seq -
4-8nts
Require Mg+
(and no ATP)
What blocks RE cleavage of DNA?
Methylation!
- Added by the cognate methylase
(Fully OR Hemimethylated) - Dam + Dcm Methylation
(Second A Or C)
No cleavage if Meth site overlaps Restriction Site
What is the primary epigenetic signal on Prokaryotes?
Methylation
What will Dpn I only cleave?
METHYLATED DNA
Sequence GATC
What will Xho I not cleave?
EUKARYOTIC methylation @ CpG islands
What will Mbo I only cleave?
NONMETHYLATED DNA
What does Ligase outcome depend on?
Conc of DNA
(Higher = InterMol + linear)
(Lower = IntraMol + circular)
What 3 activities do many DNA Polymerases have?
- Extend 3’ end (standard)
- 3’ to 5’ Exonuclease
(Proof-Reading) - 5’ to 3’ Exonuclease
(Remove DNA ahead)
What is DNA Pol PROCESSIVITY?
Amount of time the Pol stays on the DNA replicating
Why is T4 DNA Pol useful?
Trimming Fragment Ends -
Has high 3 to 5 EXONUCLEASE activity
Why is T7 DNA Pol useful?
Chain Terminator SEQUENCING
Very high processivity
Why is DNA Pol ONE useful?
Primer Removal (5 to 3)
Why is Reverse Transcriptase useful?
Used to make cDNA from RNA template
Why are thermostable polymerases useful?
Used in PCR
Taq, Vent, Pfu
What is the Kelnow fragment of DNA Pol ONE?
DNA Pol without Exonuclease activity (5 to 3)
Use to synthesis oligos for radiolabelling or Chain Terminator Sequencing
Why is T4 PN Kinase useful?
It transfers a P to the 5’ OH end
LABELS that end
(For fragments or oligo probes)
What are the FOUR properties of cloning vectors? (A MUM)
A - Autonomous Replication
M - Marker (to select for cells)
U - Unique Restriction Sites
M - Minimal nonessential DNA
(maximise cloning size range)
How long are PCR Primers?
17-25nt
What is the temperature progression in PCR?
95 > 55 > 72
What three things happen at the different temperatures in PCR?
Melt > Primer Anneal > Extension
What is PCR Error Rate like?
Greater than DNA rep
Depends on polymerase proofreading
Low error needed for cloning
How can you join ends cut by different REs??
Polymerase then Ligase
Linker ligation
Adapter ligation
Add sequences to 5’ end by PCR
What is a Linker?
A DS oligo with a restriction site
What are Adapters?
Pre-formed cohesive ends (blunt and sticky end)
Add by blunt ligation
What is a cos site?
The sites cleaved from the thread replicated from a rolling circle, which produce cohesive ends and are packaged in the phage
How do you make a Lambda insertion vector?
Remove inessential (lysogenic) genes up to 10kb and insert sequences
To increase size - stuffer region with RE sites, package genome, remove stuffer
new size up to 23kb
What is a contig?
All overlapping clones formed from chromosome walking
How do you screen PHAGE Genomic libraries?
96 well plating
OR
radioactive probes for gene
How do you screen BAC genomic libraries?
96 well plates
Or
UCSC Genome Browser
