In multi-template PCR, chimeric sequences come out from template switching during PCR reaction and mismatch repair of heteroduplexes by the host cell upon cloning. How to reduce such artifacts to avoid overestimation of haplotypes? I present here a trick to reduce such artifacts with economy use of TA cloning kit.


Incomplete extension products work as primers at the next PCR cycle to make chimeric sequences.
Inactivating enzyme in later cycles is prone to make incomplete extension.


In later PCR cycles when the reaction reaches a plateau, rennealing between PCR products making
heteroduplexes becomes predominant over annealing between PCR product/template and primer.
Cloning heteroduplex sequence into a host cell, the mismatch repair system works randomly
using either strand as template to make chimeric sequence.

Optimization of number of cycles with a real time PCR, use of heteroduplex-specific decomposition enzyme, and addition of three cycles in refreshed PCR mixture have been reported to reduce chimeric sequences upon PCR and cloning. The latter method is cheap and easy without additional machine or enzyme. Recently I tested it, and it turned out that only a single re-extension is enough for cloning reducing chimeras.


Heteroduplexes are heat denatured in a refreshed PCR mix containing sufficient primers, dNTPs and
enzyme. Primers competitively and preferentially anneal to single-strand DNAs suppressing
heteroduplex formation. Homoduplexes are subsequently recovered by a single re-extension of nascent
strand.


Outline

1st round PCR
  not more than 30 cycles (IMPORTANT)¹⁾

Electrophoresis
  to quantify PCR product

Dilute
  to c.a. 10ng/uL with MQ²⁾

Single re-extension
  1uL diluted PCR product
  + 9uL PCR mix
    {dNTPs (Takara) 0.8uL, 10x ExTaq buffer 1uL, 1/10x ExTaq 0.5uL,
    F-primer (5pM/uL) 1uL, R-primer (5pM/uL) 1uL, MQ 5.2uL}
      1/10x ExTaq =
        {ExTaq (5U/uL) diluted 10 times with 10mg/mL BSE (Boehringer #238040)}
  {94°C (180sec) -> X°C (30sec) -> 72°C (30min) -> 4°C} x 1

Ligation
  1uL re-extension product
  + 1uL 1/10x pGEM-t {pGEM-t (Promega) diluted 10 times with MQ}³⁾
  + 3uL ligation mix {2x buffer 2.5uL, ligase 0.5uL (pGEM-t kit)}
    IMPORTANT; add ligation mix finally⁴⁾
  15°C 4hrs -> 4°C several hrs (optional)
  + 5uL TE

TF
  Put competent DH5alpha on ice to thaw⁵⁾
  4uL of the diluted ligation product in 2ml Eppendorf tube on ice
  Pour 50uL of DH5alpha onto the ligation product
  On ice 15min
  44°C 45sec
  On ice 1min (immediately prepare the next step)
  +500uL SOC (37°C)
  Rotate 10min (if SOC is chilled, 15min)
  Spread 100uL on an LB amp+ X-gal+ plate
  37°C o/n

Efficiency
  100-500 colonies, more than 1/2 white

An example from a tetraploid loach (RAG1, 1.5kb, 4 alleles
with 0.9 - 3.8% nt differences)⁶⁾
----------------------------------------------------------
reaction condition        cycles      chimera/total clones
----------------------------------------------------------
no re-extension            35            22/25
no re-extension            30             5/16*
single re-extension        35            17/28
single re-extension        30             3/19
----------------------------------------------------------
* An allele out of four was not recovered.

REFERENCE
Saitoh K, Chen WJ. 2008. Reduction of cloning artifacts for recovery of allelic sequences by T7 endonuclease I cleavage and single re-extension of PCR products -- A benchmark. Gene 423:92-95.

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<Note>
1) Reaction with ExTaq (Takara). Many chimeras appeared even upon re-extension from PCR product of 35 cycles.
2) The value is in case of 1.5kb insert. Adjust DNA amount according to length of the insert.
3) This combination makes a good insert-vector ratio of about 2ng of insert (1.5kb) and 5ng vector (3kb).
4) When there are a number of inserts to ligate, mix insert and vector first, and then add ligation enzyme mix later. Frequent self-ligation occurs if mixture of enzyme and vector is added to insert (mixing enzyme and vector first), though it is easier.
5) Home-made competent cell.
6) Chimeras cannot be completely eliminated even upon single re-extension. Use the results cautionarily.

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