WHY SNaPshot

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WHY SNaPshot??
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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.

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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.

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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

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ELECTROPHEROGRAM OF FOUR MULTIPLEXED SNPs IN
ABCG2--SHOWING HOMOZYGOTES
BLUEG GREENA REDT BLACKC ORANGE STD
40G/G
45G/G
50T/T
35T/T
STD
STD
BLUEG GREENA REDT BLACKC ORANGE STD
40A/A
45A/A
35C/C
STD
50C/C
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.
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ELECTROPHEROGRAM OF FOUR MULTIPLEXED SNPs IN
ABCG2 -- SHOWING HETEROZYGOTES
BLUEG GREENA REDT BLACKC ORANGE STD
G
G
T
T
STD
STD
C
C
A
A
BLUEG GREENA REDT BLACKC ORANGE STD
G
G
T
T
STD
C
C
A
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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