Kun:LabNotes/Haplotyping/2009-2-12

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Comparison of MDA using N6, LN6 and N9 primers[edit]

  • The objective of this experiment is to compare the amplification bias of several variants of MDA protocols:
    • Control: N6 primer: 30C
    • LN6 (LNA primer): 30C, 37C, 30C + trehalose
    • N9: 30C + trehalose
  • The template DNA is GM20431, 30pg/reaction (5 copies of diploid genome)
  • Two replicates of GM20431 and NTC (20 reactions in total)

Prepare Alkaline Lysis Solution (ALS) and Neutralization Solution (NS)[edit]

  • ALS: (400mM KOH, 100mM DTT, 10mM EDTA)
    • Prepare 5M KOH: weigh one KOH pallet, add nuclease free H2O to make 5M KOH. Make fresh solution every week.
    • Mix 8ul 5M KOH, 10ul 1M DTT, 102ul nuclease free H2O.
  • NS buffer (0.4M HCl, 0.6M Tris.HCl pH 7.5):

Set up MDA reactions[edit]

  • Perform serial dilution of GM20431 gDNA to 30pg/ul.
  • Perform UV treatment of ALS and NS buffer 10ul/tube for 5 min;
  • Prepare 2X SYBR Green I solution from the 50X stock solution;
  • Mix 1ul template with 1.5ul ALS buffer, RT for 3min;
  • Transfer to cool block;
  • Add 1.5ul NS buffer;
  • Add 16ul Master mix:
                                  x13           x9
                                 Tre(-)        Tre(+)
  Trehalose(1.2M)                  0ul         5.0ul
  10x RepliPhi phi-29 buffer     2.0ul         2.0ul
  1mM primer                     1.0ul         1.0ul
  25mM dNTP                      0.8ul         0.8ul
  2X SYBR Green I                1.0ul         1.0ul
  H2O                            9.7ul         4.7ul
  RepliPhi Phi-29 (100U/ul)      1.0ul         1.0ul
  • Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.
  File:2009-02-12-MDA-N6.png File:2009-02-12-MDA-LN6.png
  File:2009-02-12-MDA-LN6-Tre.png File:2009-02-12-MDA-N9-Tre.png
  File:2009-02-12-MDA-LN6-37C.png

Illumina Infinium Data[edit]

GM20431 Correct Calls LOH Incorrect Calls No Calls
LN6.GType 186950 56700 56735 249883
N6.GType 230134 51403 45395 223336
N9+Tre.GType 335118 54811 48774 111565
30pg.GType 303840 52613 47240 146575
LN6+Tre.GType 299575 56480 50434 143779

Relative intensity compared with 200ng gDNA

Amplicon GM20431 N6.GType GM20431 30 pg.GType GM20431 LN6.GType GM20431 LN6+Tre.GType GM18507 200 ng.GType GM18507 30pg.GType GM20431 N9+Tre.GType
STDEV 1.069 0.975 1.391 0.904 0.208 0.889 0.835
Mean -0.326 -0.124 -0.336 -0.214 0.018 -0.487 -0.160


It looks like the amplicon using the N9 primer + Trehalose has the most number of correct calls.