SUBCLONING I

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SUBCLONINGPart 2
Sarah L. Donahue, PhD Research Fellow H. Earl
Ruley Lab MCN AA-4210
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Overview of DNA Subcloning

Vector
DNA of Interest
Recombinant Vector
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Ideal Cloning Reaction

• A) Digest insert with
• Bam HI and Xho I
• B) Digest Plasmid with Bam HI and Xho I
• Gel purify fragments
• 3. Ligate fragments

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• Complex cloning reactions
• Cloning Kits
• -T/A cloning Kit
• -PCR script cloning Kit
• Fusion proteins

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How to Deal with Non-ideal or non-existent
restriction sites
What if You Cant Find Suitable Restriction
Sites?

• 1. Cleavage with REs that produce compatible
  overhangs
• 2. Cleavage with two RE that produce blunt ends
• Cleavage by RE followed by fill-in of 5 overhang
  to generate blunt ends (klenow)
• 4. Use PCR to introduce the correct REs

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Complex cloning reaction
Mfe I
Hind III
Cla I
Pst I
Sma I
Bam HI
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• 1. Compatible cohesive ends
• Eco RI 5G?AATTC3 G
  5..AATTC
• 3CTTAA?G5 CTTAA...5
  G
• Mfe I 5C?AATTG3 C 5..AATTG
• 3GTTAA?C5 G TTAA...5
  C

These two enzymes are not isoschizomers!
However, the cuts made by the enzymes create
overhangs that are complementary.
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Compatible Cohesive Ends

• Digest insert with
• Mfe I and Bam HI.
• 2. Digest Plasmid with Eco RI and Bam HI.
• Gel purify fragments.

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Tables of Compatible Ends
New England Biolabs catalog
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• 2. Blunt-end ligation
• Sma I 5CCC-OH P-GGG3
• 3GGG-P HO-CCC5
• Dra I 5TTT-OH P-AAA3
• 3AAA-P HO-TTT5
• Ligation Reaction 5CCCAAA3
• 3 GGGTTT5

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• 3. Cut DNA with restriction enzyme and fill-in or
  remove overhangs with Klenow fragment
• Proteolytic product of E. coli Polymerase I which
  removes
• 3 overhanging DNA and fills-in 5 overhanging
  DNA.
• Bam HI 5G?GATCC3 G
  5GATCC
• (5 Overhang) 3CCTAG?G5 CCTAG5
  G
• Pvu I 5CGAT?CG3 CGAT3 CG
• (3 Overhang) 3GC?TAGC5 GC 3TAGC
• Blunt Ligation reaction 5GGATCCG3
• 3CCTAGGC5

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• 3. Cut DNA with restriction enzyme and fill-in or
  remove overhangs with Klenow fragment
• Proteolytic product of E. coli Polymerase I which
  removes
• 3 overhanging DNA and fills-in 5 overhanging
  DNA.
• Bam HI 5G?GATCC3 G
  5GATCC
• (5 Overhang) 3CCTAG?G5 CCTAG5
  G
• Pvu I 5CGAT?CG3 CG3 CG
• (3 Overhang) 3GC?TAGC5 GC
  3GC
• Blunt Ligation reaction 5GGATCCG3
• 3CCTAGGC5

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4. PCR-based cloning

• Incorporate restriction enzyme sites in primer
  sequence
• This can mean long primer sequences.
• Extra bases are required for the PCR product to
  be digested by the desired restriction enzyme.

BamH I GGATCC
GCGGCCGC Not I
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4. PCR-based cloning
BamH I cgGGATCC
Not I GCGGCCGCtaaactat
Cleavage 2 Hr
Bam HI cGGATCCg 10
cgGGATCCcg 90
Not I ttGCGGCCGCaa 10
ataagaatGCGGCCGCtaaactat 25
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PCR Cloning Kits

• T/A Cloning Kit (Invitrogen)
• Taq polymerase has a nontemplate-dependant
  transferase activity that adds a single
  deoxyadenosine (A) to the 3 end of PCR products.
  The linearized vector supplied in the kit has
  single, overhanging 3 deoxythymide (T) residues.
• Disadvantage is Taq polymerase has a high error
  rate.

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PCR Cloning Kits

• PCR-Script Cloning Kit (Statagene)
• For use with blunt end PCR products generated
  with Pfu DNA polymerase. The PCR product is
  incubated with a linearized vector DNA. The
  efficiency of ligation is increased by the
  addition of the Srf I restriction enzyme which
  cleaves any unligated vector.

Srf I is a novel rare-cleavage restiction enzyme
that recognizes the sequence 5-GCCC?GGGC-3
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(No Transcript)
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Fusion Proteins

• Add a specific epitope (tag) to either the
  N-terminus or C-terminus of your gene of interest

The Peptide sequence of the FLAG epitope ATG
GAC TAC AAA GAC GAT GAC GAC AAG CTC GAG M
D T K D D D D
K Xho I
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Amino Acid reading frames

• cDNA sequence of TEL
• CTC GAG ATG TCC AAT TTA
• Xho I M S N L
• Flag Vector
• ATG GAC TAC AAA GAC GAT GAC GAC AAG CTC GAG
• M D T K D D D
  D K Xho I
• FLAG-Tagged TEL
• ATG GAC TAC AAA GAC GAT GAC GAC AAG CTC GAG ATG
  TCC AAT
• M D T K D D D
  D D L E M S L

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Alternative reading frames

• cDNA sequence of TEL gene
• CTC GAG ATG TCC AAT TTA
• Xho I M S N L
• Flag Vector
• ATG GAC TAC AAA GAC GAT GAC GAC AAG GAT CCT CGA
  G
• M D T K D D D
  D K DpnI Xho I
• Nonsense protein
• ATG GAC TAC AAA GAC GAT GAC GAC AAG GAT CCT CGA
  GAT GTC CAA TTT A
• M D T K D D D
  D D D P R D V
  P T

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Vectors are available in all 3 reading frames

• Flag Vector-1
• ATG GAC TAC AAA GAC GAT GAC GAC AAG GAT CCT CGA
  G
• M D T K D D D
  D K DpnI Xho I
• Flag Vector-2
• ATG GAC TAC AAA GAC GAT GAC GAC AAG GAT CTC TCG
  AG
• M D T K D D D
  D K DpnI Xho I
• Flag Vector-3
• ATG GAC TAC AAA GAC GAT GAC GAC AAG GAT CTA CTC
  GAG
• M D T K D D D
  D K DpnI Xho I