Dahlquist:Lactase Persistence Genotyping by qPCR

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This protocol is based off of Weinlander, K. M., Hall, D. J., & De Stasio, E. A. (2010). RFLP analysis and allelic discrimination with real‐time PCR using the human lactase persistence trait. Biochemistry and Molecular Biology Education, 38(3), 167-171. doi: 10.1002/bmb.20357 (full text)

Primers

Forward primer sequence:

5'-GAGTGTAGTTGTTAGACGGAGAC-3'

qPCR primer sequence for T genotype:

5'-AGGCCAGGGACTACATTATC-3'

qPCR primer sequence for C genotype:

5'-AGGCCAGGGGCTACATTATC-3'

Weinlander qPCR primers aligned-to-448bp-product.jpg

Reaction Mix

From Weinlander paper:

7 ng genomic DNA
1.4 µL of 3.5 µM forward primer
1.4 µL of 3.5 µM reverse primer C or T
0.5 µL 50X ROX
12.5 µL master mix
Water to 25 µL

Note: the final concentration of each primer is 0.2 μM and the master mix contains modified Thermus brockianus DNA polymerase, dNTPs, SYBR Green, MgCl2, and buffer solution.

Modified from Apex qPCR GREEN Master Mix Manual:

Component                     Vol./reaction*             Final concentration*
Apex qPCR 2X Master Mix       12.5 μL                     1X
Primer A (10 μM)               0.5 μL (0.125 – 1.25 μL)   0.2 μM (0.05 – 0.5 μM)**
Primer B (10 μM)               0.5 μL (0.125 – 1.25 μL)   0.2 μM (0.05 – 0.5 μM)**
PCR-grade H2O                  6.5 μL                     ---
Template DNA                   5   μL                     genomic DNA: 20 ng (1 – 100 ng) or plasmid DNA: 0.5 ng (0.1 – 1 ng)
TOTAL volume                  25   μL                     ---

* Suggested starting conditions; theoretically used conditions in brackets.
** Optimization of primer concentrations is highly recommended. Note that I had to change the final concentration to 0.2 μM from 0.1 μM because manual incorrect for the 0.5 μL volume. I changed the theoretical volumes of the primer to match the theoretical final concentrations.

Organizing Reactions

  • Each sample (template) needs to be run with two different primer pairs, which means two master mixes will be made, one for each primer pair:
    • Forward + Reverse (C)
    • Forward + Reverse (T)
  • Each sample + primer pair needs to be run in duplicate or triplicate as technical replicates to account for technical differences (pipetting error, etc.)
  • There needs to be a negative no DNA template control (using water instead of DNA) for each primer pair/master mix
  • We will use the plasmids as positive controls, "T" plasmid, "C" plasmid, and a mix of "T" and "C" plasmid. Each of these is run with both primer pairs.
    • We should do a dilution series of 3-5 dilutions for each of these to determine the right concentration of plasmid template to use going forward.

Instrument Protocol

qPCR carried out in four stages run consecutively.

  1. Stage 1: 50.0°C for 2 minutes.
  2. Stage 2: 95.0°C for 10 minutes.
  3. Stage 3: 40 cycles of 95.0°C (for 15 seconds) and 60.0°C (for 1 minute).

Results for each sample were determined by selecting only the amplification plots of both tubes after the threshold was set.

Troubleshooting

  • Sigma-Aldrich qPCR Guide
  • Their recommended standard instrument protocol is:
    1. Initial denaturation 94°C for 2 min
    2. Denaturation 94°C for 15 sec
    3. Annealing, extension, and read fluorescence 60°C or 5°C below lowest primer TM for 1 min
    4. repeat steps 2 and 3 for 40 cycles
  • has diagram regarding how SYBR works
  • explains quantification cycle relative to amount of starting material (i.e. dilutions)
  • lists supplies needed and components of assay and why each is important
  • has troubleshooting guide