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Genotyping
Services
Functional Genomics Laboratory
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Research
Scientist Pat Janssen sets up a Taqman-based genotyping assay.
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The
Functional Genomics Core Laboratory uses an ABI PRISM 7700 Sequence
Detection System, fluorogenic TaqMan™ probes, and specific
PCR primers to carry out 5’-nuclease-based genotyping (allelic
discrimination) assays. Since 2000, the Function Genomics Laboratory
has developed over 100 TaqMan-based genotyping assays and identified
over 40,000 genotypes. This core facility will utilize a similar
approach to develop any new genotyping assays.
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Research
Technologist Hannah Viernes sets up
96-sample thermocycling reactions for a PCR-based genotyping assay.
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TaqMan™ Detection System uses fluorogenic probes to detect
specific nucleic acid sequences and single nucleotide polymorphisms
for allelic discrimination by using the 5’ nuclease assay.
The use of differentially labeled fluorogenic probes in the 5’
nuclease assay combines PCR amplification and detection into a single
step eliminating the need for post-PCR processing. In this system,
a hybridization probe included in the PCR is cleaved by the 5’
nuclease activity of Taq DNA polymerase only if the there is perfect
sequence complementarity between probe and target sequence. Since
each allele specific probe consists of an oligonucleotide labeled
both with a 5’ reporter dye (each probe labeled with a different
reporter dye) and a 3’ quencher dye, cleavage of the probe
liberates the reporter dye, which causes an increase in its fluorescence
intensity. A mismatch between probe and target substantially reduces
the efficiency of hybridization and cleavage. Therefore, a mismatched
probe does not attribute appreciably to the final fluorescent signal.
Additionally, since fluorescence signal is generated only if the
target sequence for the probe is amplified by PCR, no signal is
generated by non-specific amplification. Thus, substantial increase
in fluorescent signal for just one of the reporter dyes indicates
homozygosity for the corresponding probe specific allele and an
increase in both signals indicates heterozygosity. |
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Contact
Federico
Farin, Core Manager
freddy@u.washington.edu, (206) 685-7285
UW Box 354695
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