Submitted to: Plant and Animal Genome
Publication Type: Abstract Only
Publication Acceptance Date: 1/10/2004
Publication Date: 1/14/2004
Citation: Xin, Z., Chen, J., Burke, J.J. 2004. A low cost high efficient method for genetic mapping using indel markers[abstract]. Plant and Animal Genome. Paper No. 227.
Technical Abstract: Insertion/Deletion (InDel) markers are an important class of molecular markers for mapping heritable traits in animals and plants because the markers with large insertions or deletions (>10 bp) can be directly scored on agarose gels. The advent of fragment analysis with fluorescence-tagged primers made this class of markers especially useful since a single bp insertion or deletion can be reliably resolved by electrophoresis on acrylamide gels or capillary columns. In conventional fragment analysis, one primer has to be tagged with a fluorescent phosphoramidite for each marker. We have improved fragment analysis by labeling the PCR fragment with a universal primer tagged with a fluorochrome. ABI forward sequencing primer (an improved version of M13F) was tagged with FAM, HEX, or NED; the same universal primer sequence was fused to the 5' end of the forward primer for each marker. PCR product was labeled fluorescent by including 50 nM fluorochrome-tagged universal primer in the PCR reaction mix. No special PCR condition was required; the conditions suitable for the none-tagged primers were fully compatible with the inclusion of tagged universal primer. Less than 0.1 micro liter of PCR product was sufficient for fragment analysis without further purification. PCR products of the markers that differed either in size or color could be pooled into one run. Moreover, this method was fully compatible with our high throughput DNA preparation method (Biotechnique 34:820-826) and greatly reduced the cost associated with map-based cloning.