High-Throughput SNP Genotyping by SBE/SBH Ion Mandoiu and Claudia Prajescu, CSE Department, University of Connecticut

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High-Throughput SNP Genotyping by SBE/SBHIon
Mandoiu and Claudia Prajescu, CSE Department,
University of Connecticut
SBE/SBH Assay Steps
TTGC

• Synthesize primers complementing the upstream
  sequence of target SNP loci on either sense or
  antisense strands

GATA

• Mix primers with solution of PCR amplified
  genomic DNA containing target SNP loci

T

A
T

• Rise temperature to denature double stranded
  genomic DNA then reduce temperature to allow
  primers to hybridize. Hybridization conditions
  should be stringent enough to prevent primer
  hybridization at locations other than SNP loci

T
A
T

• Add DNA polymerase and fluorescently labeled
  dideoxynucleotides (ddNTP) to the solution.
  Primers are extended by single ddNTPs
  complementing SNP values. This can be run as one
  reaction with ddNTPs labeled by 4 distinct
  colors, or as 4 independent reactions each of
  which uses only one type of ddNTP.

TTGCA
T
A
GATAA
T

• Primers are separated from genomic DNA and
  hybridized to an array containing all k-mers for
  k8-10. For more reliable solid-phase
  hybridization, the array can alternatively
  contain a set of isothermic probes (i.e., probes
  that have similar melting temperatures), such as
  the set of all c-tokens for c11-13 Ben-Dor et
  al. 00

AA AC CC CA
AT AG CG CT
TT TG GG GT
TA TC GC GA

• The array hybridization pattern is analyzed to
  make the genotype calls. To enable unique
  decoding, each primer should hybridize to at
  least one array probe to which no other primer
  hybridizes