Subcloning

1
Subcloning

• Michael W. Thompson, Ph.D.
• IGP Methodology
• October 9, 2003
• michael.w.thompson_at_vanderbilt.edu

2
Subcloning Why Bother?

• Subcloning refers to movement of a piece of DNA
  from one vector to another
• Transfer gene of interest to expression vector
• Shotgun sequencing
• Transfer gene to control of a different promoter
• Fusion proteins
• Characterization of novel cDNAs

3
Subcloning Basics

• Choose suitable vector
• Identification of suitable restriction sites for
  gene transfer
• Digestion of insert and vector with restriction
  enzymes
• Purification of insert and/or vector (if
  necessary)
• Ligation of DNA fragments with T4 DNA ligase
• Transformation of ligation products
• Selection of recombinant DNA molecules

4
Choosing a Vector

• Appropriate vector for organism or biological
  system used
• Appropriate selection method
• MCS compatible with restriction sites of insert
• Other vector features
• affinity tags
• promoters
• stop codons!!!

5
Which Vector Would You Pick?
The Task
You wish to express YFG protein in E. coli
YFG
EcoRI
SalI
XmaI
EcoRV
ClaI
HindIII
Vector B
Vector A
6
Choosing Restriction Sites

• Commercially available enzymes
• Unique restriction sites preferred
• Considerations
• Restriction enzyme characteristics
• Proximity to ORF if expressing protein
• Preservation of reading frame
• Considerations for screening products

7
What if You Cant Find Suitable Restriction Sites?

• Blunt-end ligation
• Can be done by restriction enzyme or
  removal/fill-in of overhang
• Cut DNA with restriction enzymes that leave blunt
  ends (SmaI, EcoRV)
• Cut DNA with restriction enzyme and fill in or
  remove overhangs with Klenow fragment
• Fill in with Klenow fragment
• 5 overhang will be filled in (add dNTPs)
• 3 overhang will be removed
• Heat inactivate Klenow
• Ligate to similarly treated vector
• Lowering ligation temperature (12-14oC), more
  ligase, and/or additives (PEG) may enhance
  ligation

8
Preparing DNA for Ligation
Vector
Insert
1-2 mg DNA, digest with restriction enzyme(s)
3-6 mg DNA, digest with restriction enzyme(s)
Heat inactivate enzyme(s)
Purify insert from low-melt gel, or may remove
short polylinker fragments by spin column or
ethanol precipitation (how?)
Remove 5 phosphate group (why?) Inactivate
phosphatase purify by
gel or spin column (why?)
9
Why Purify DNA for Ligation?
10
Why Remove 5 Phosphates?

• Prevents re-ligation of vector
• Essential when blunt-end cloning or using a
  single restriction site on the vector where it
  may re-anneal

GAATTC CTTAAG
G
AATTC
CTTAA
G
Overhangs re-anneal
EcoRI
Cut with EcoRI
GREAT Ligase Substrate!!!
11
Removal of 5 Phosphate

• Prevents re-ligation of vector
• Essential when blunt-end cloning or using a
  single restriction site on the vector where it
  may re-anneal

OH
HO
G
GAATTC CTTAAG
AATTC
CTTAA
G
OH
HO
EcoRI
G
AATTC
CTTAA
Not a suitable T4 ligase substrate
G
12
Why Remove 5 Phosphates?
13
Performing a Ligation

• Mix together purified cut vector and insert
• X ng/ml vector
• 2 to 3 X ng/ml insert (why?)
• 10X Ligase buffer (to 1X, containing dATP, 1mM in
  rxn)
• 0.5-1 ml T4 DNA Ligase
• H2O to volume
• Incubate 1-16 hours at 14-16oC
• Do we need any controls?

14
Performing a Ligation

• Transform the ligation into E. coli, plate on
  selective media
• chemically competent cells
• Electroporation (ligation must be precipitated)
• Pick colonies, isolate DNA from minipreps
• Screen clones by restriction digest or by DNA
  sequencing to ensure correct reading frame

15
Screening Recombinants

• When performing non-directional subcloning, need
  effective means of determining orientation of
  insert
• Use unique restriction site(s) within the vector
  and/or insert and screening by restriction digest
  is preferred to sequencing

16
How Would You Determine Orientation of the Insert?
EcoRI
BamHI
HindIII
1.6kb
3.3kb
0.5kb
EcoRI
EcoRI
BamHI
5.4kb

EcoRI
EcoRI
HindIII
3.3kb
1.6kb
0.5kb
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Some Things to Consider

• Clean, purified DNA fragments work better
• Less DNA manipulation before ligation is better
• Directional, cohesive-end ligations are easier,
  if possible with given system
• Use a 21 insertvector ratio (sometimes higher
  for blunt-end ligation)
• Blunt-end ligations may be enhanced by lowering
  the dATP (0.1 mM), adding more ligase, and/or
  using 5 PEG-4000 (a crowding agent)
• When cloning into a single site or blunt-end
  cloning, removal of 5 phosphates from vector is
  essential

18
Stupid Cloning Tricks!

• Using PCR to facilitate subcloning

EcoRI
BamHI
PCR
BamHI
EcoRI
19
Subcloning by PCRConsiderations

• Adding extra nt beyond engineered restriction
  site may improve restriction enzyme digestion
  efficiency
• Primers will be long!!!
• Make the matching portion of the primer as long
  as possible
• Adding extra nt between new site and insert can
  be used to shift reading frame for insertion into
  vectors where this is a problem
• May require some tweaking of PCR conditions (low
  annealing temp, longer annealing time, more
  cycles, higher Mg2)
• Use of proofreading enzyme (Pfu, Pfx, Vent) may
  help reduce spurious mutations
• Sequencing will be necessary!!!

20
In-Class Subcloning Exercise

• Devise a strategy to subclone the given cDNA into
  the indicated vector
• Exercise A Your boss is a cheapskate. Subclone
  MP100 into any of the four available expression
  vectors. Bonus points if you can get it into one
  with an affinity tag!
• Exercise B You have any and all materials at
  your disposal, but you MUST clone MP100 into the
  given expression vector, and it MUST be in frame
  with the affinity tag!