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Temperature Gradient Capillary Electrophoresis (TGCE):
TGCE is a method of rapidly detecting heteroduplexes formed between
two PCR products differing by as little as a single nucleotide (we have
detected a SNP in an 841 nt fragment and a 1-bp IDP in an 800 bp fragment)
(Hsia
et al., 2005). Two identical sequences result in a single TGCE peak;
non-identical sequences give multiple peaks.
Please click on the thumbnail at right to view a larger
image.
Background: High temperature overcomes the hydrogen bonds that hold
the two strands of DNA together causing the normally double-stranded (ds)
DNA to become single stranded. Lowering the temperature allows re-formation
of double strands (renaturation or reannealing). When two single-stranded
sequences of DNA that are similar but not exactly identical are allowed
to renature, the dsDNA that forms is termed a heteroduplex (versus homoduplex).
Sequence differences can be due to changes in the identity of one or more
nucleotides (SNPs or single nucleotide polymorphisms) or an insertion or
deletion of one or more nucleotides (IDPs – insertion/deletion polymorphisms)
or more complex changes.
TGCE is a method of rapidly detecting heteroduplexes formed between two PCR products differing by as little as a single nucleotide (we have detected a SNP in an 841 nt fragment and a 1-bp IDP in an 800 bp fragment) (Hsia et al., unpublished). Two identical sequences result in a single TGCE peak; non-identical sequences give multiple peaks.
TGCE Concept: Li Q, Liu Z, Monroe H, Culiat CT (2002) Integrated platform
for detection of DNA sequence variants using capillary array electrophoresis.
Electrophoresis 23:1499-1511. For more information on using TGCE
for genotyping click here.
Determination of the genome of a recombinant inbred via TGCE
Click on the thumbnail for a larger image.
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Sequence
of A1 alleles from B73 and Mo 17
Click
on the thumbnail for a larger image. A genetic map that will contain 4,500
new markers that differentiate B73 and Mo17 is being produced. This
project is facilitated by a throughput of six to twelve 96-well plates
per day requiring little operator intervention. |
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