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Genotyping by SNaPshot Multiplex

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Genotyping by SNaPshot Multiplex Powered By Docstoc
					WHY SNaPshot??


                 1
  Genotyping by SNaPshot
         Multiplex
• Genotyping method based on a single-
base extension, that is, each probe binds
to complementary DNA template in the
presence of fluorescently labeled ddNTPs
and AmpliTaq DNA Polymerase.
Polymerase extends probe by one base,
adding one ddNTP to 3’ end.

• Separation of nucleotide peaks occurs
on ABI instrument.
                                          2
  Preparatory Work for SNaPshot
       Multiplexing of SNPs
Design PCR Primers and Probes
Specifications of PCR Primers:
19-22mers
Use low concn in PCR reaction: 0.2 - 0.4 um

Specifications of Probes [to identify different SNPs]:
Minimum Length = 20/25 bp
Maximum Length = 75 bp
Best resolution obtained within 25-75 bp, probes 5 bp apart to
separate peaks well.
                                                                 3
         METHOD PROTOCOL
Step 1 - Individual PCR reactions for each variant, 6.0
ng/ul DNA minimum, check on gel [approx 5-6 hrs]

Step 2 - EXO-SAP Clean-up PCR reactions- pool 20% each
PCR [approx 2 hrs]

Step 3 - Perform Multiplex SNaPshot extension reaction
with pooled purified PCR products and pooled probes
[approx 2-3 hrs]
Step 4 - Post-extension SAP clean-up [approx 2 hrs]

Step 5 - Run samples with ctrls
ON ABI 3130, approximately 4 hours/96 reaction plate
                                                          4
  ELECTROPHEROGRAM OF FOUR MULTIPLEXED SNPs IN ABCG2--
                SHOWING HOMOZYGOTES


     BLUE=G
     GREEN=A
     RED=T                         40G/G
     BLACK=C
                                                  45G/G
     ORANGE =STD
                         35T/T                                       50T/T
    STD                                                                                  STD




    BLUE=G
    GREEN=A                         40A/A
    RED=T                                              45A/A
    BLACK=C
    ORANGE =STD      35C/C
                                                                  50C/C                  STD
       STD


Note: Because of influence of dye on mobility shift of the DNA fragments, reported sizes differ
by about 5 bases from actual sizes of probes.                                                     5
ELECTROPHEROGRAM OF FOUR MULTIPLEXED SNPs IN ABCG2 --
             SHOWING HETEROZYGOTES


 BLUE=G
 GREEN=A               G
 RED=T
 BLACK=C                     G
 ORANGE =STD
                   T                      T
  STD                                            STD
               C                      C
                       A          A




 BLUE=G
 GREEN=A               G
 RED=T
 BLACK=C                      G
 ORANGE =STD       T                      T
                                                 STD
               C
                       A              C
    STD                           A

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        Genotyping by SNaPshot
               Multiplex
ADVANTAGE:
• Can genotype up to 10 variants simultaneously
[Genotyping of 17 SNPs w/overlap reported, Quintans et al., Forensic Science
International 140 (2004) 251-257.]


DISADVANTAGES:
• Multi-step method - higher turnaround time
• Cost = Approx $3.84 per reaction
          Approx 0.96 per SNP [4 variant multiplex]
         Approx 0.38 per SNP [10 variant multiplex]
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posted:7/23/2013
language:English
pages:7