Cloning Flashcards
What are the uses of cloning?
isolate, propagation of dna fragments
in sequencing
for preparing probes
expression & purification of large protein quantities
invitro mods of DNA
What are the basic steps of cloning?
Gen. of dna fragments by REs
ligation
transformation
propagation
isolation & screening
What are the methods to generate DNA fragments?
chromosomal dna - RE digestion / PCR RNA -- cDNA = eukary recombinant dna - subcloning chemical synthesis PCR-amplified DNA mechanical shearing
Compare RE cleavage and PCR
RE - several fragments result – fragment isolation – low melting agarose, dialysis tubing
PCR - RT PCR
On what factors the selection of vector depends on?
Purpose - cloning - based on selection markers or expression - specialized for host?
Insert size
vector size
res. sites present in its MCS
ability to screen for inserts
copy number
efficiency
Why sticky ends are easier to ligate?
ss overhangs that are complementary can form H bonds than ligase form phosphodiester bonds
blunt end combining need energy
How to minimize self-ligation?
using alkaline phosphotase -OH, -OH > Ligase - join OH-P ends But nicks present -OH -OH but not repaired here after transformation the host replication process repairs the nicks
List the strategies used to obtain compatible ends
RE digestion - if diff RE were used > to make compatible > Linkers Adapters homopolymer tailing PCR products TA cloning
What does a linker do? Show the steps
make blunt – sticky
DNA fragment + linker mol.s + T4 dna ligase + ATP –> +Eco RI –> sticky ends + vector
What is an adapter? properties? steps?
foreign dna + adapter(one end dephosphorylated) + T4 dna ligase + ATP –> polynucleotide kinase + ATP –> sticky ends + vector
Show the steps for homopolymer tailing. why a exonuclease is used?
dna fragment, vector + L-exonuclease > 5’-3’ exonuclease –> dna fragment + terminal transferase + dATP & vector + dTTP + terminal transferase –> mix & anneal
Which determines the no. of nt added in homopolymer tailing
conc of terminal transferase
time
How to use PCR products to get compatible DNA fragments?
Primers designed with RSS & PCR optimized to anneal primers carefully
denature > anneal primers > PCR amplification > res. digestion
What is done in TA cloning?
No RSS
T & A added to vector & fragment
insert is created by PCR – 3’ added with A overhangs – best if pcr primers have 5’-G – max possibility of taq pol to add A overhangs
Vectors comm. available having T overhangs - TOPO vector
How topoisomerase I work?
topo I recognize CCCTT on ds dna and cut 1 strand at the last T. the energy from this broken phosphodiester –> 3’-P + Tyr of topo I –> after enzyme reforms that bond bw nucleotides
