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Human CHD2 (long isoform) Difficult PCR Amplification

PCR Success Story #5

Project Description

Difficult PCR Amplification of CHD2 (long isoform) from human cDNA

This project looked pretty simple but it ended up being a HUGE challenge and very Difficult PCR to achieve. The constraits were caused by primer design and the target’s sequence. The customer mentionned that all other high-fidelity DNA polymerases failed at amplifying full lenght CHD2 long isoform from human lymphocyte cDNA, The researcher provided us with PCR primers and cDNA.

Difficult PCR Challenges: Very long forward primer, very low GC content of the gene-specific target annealing sites and no 3′ end GC-clamp. The sequences in bold letters are gene-specific:

CHD2 long isoform (5222 kb with extensions)

  • Reverse: CGCGAATTCTTATGTTTTCCGAACATTCCAGTT (gene-specific is 33% GC and Tm = 52°C)

Project Details

Client: CR-CHUM
Date: April 25th, 2016
Type of experiment: High-Fidelity amplification from cDNA
DNA Polymerases: TransStart FastPfu and KD Plus

Competitors: None

Difficult PCR Amplification of CHD2

Amplification of CHD2

This was the most challenging PCR we ever attempted. Nevertheless, we successfully amplified full-lenght long isoform of human CHD2 using TransStart FastPfu and KD Plus. The amplified fragment was confirmed by sequencing.

PCR reaction setup:

  • H2O : 12.4 ul
  • 5x buffer : 4 ul
  • dNTPs (2.5mM): 1.6 ul (0.2 mM final)
  • F1 (10 uM): 0.4 ul (200 nM final)
  • F2 (10 uM): 0.4 ul
  • cDNA : 0.8 ul human cDNA
  • Pol (2.5 u/ul for FastPfu; 1u/ul for KD Plus) : 0.4 ul

PCR cycling :

  1. Denaturation: 120s at 95 °C
  2. 30 x
    1. Denaturation: 20s at 95 °C
    2. Annealing: 20s at 53 °C
    3. Extension: 70s at 68 °C
  3. Final extension: 200s at 68 °C

Problems with AT- or GC-rich PCR?