Modification of the GeneScan 2500 fluorescent dye standard for accurate product sizing.
This article describes a procedure for modification of the commercially prepared GeneScan 2500 size standard for allelotyping with large DNA fragments such as variable number tandem repeats (VNTRs). Here a procedure was used to adapt commercially available size standards for the sizing of the interleukin-6 (IL6) 3'VNTR, which has allele sizes ranging from 600 to 900 base pairs. The procedure involves inclusion of products from the target PCR reaction as additional size standards for use with the size standard and is therefore applicable to the sizing of any product with any commercial size standard. Initially alleles were sized by fluorescent cycle sequencing to give a true estimate of their size (base pairs). Subsequently, the major alleles were labeled with a pig-tailed ROX-dye-labeled primer and inserted into the standard range. Finally alleles were resized following PCR with an un-pig-tailed HEX labeled primer and the modified standard. Once the size standard is modified, stocks can be stored indefinitely and replenished by re-PCR of selected alleles using the ROX-labeled primer. Modification of this approach can be applied to the sizing of any product where it is thought that commercially available size standards are performing suboptimally. Modification of size standards in this way does not affect performance in size regions other than those for which the extra products are included.
Bartlett
J M
JM
Department of Haematology, Edinburgh Royal Infirmary, Scotland, UK. Allelotype@DNASHEF.com
Crilly
A
A
White
A
A
Madhok
R
R
D000483:Alleles; D000818:Animals; D005456:Fluorescent Dyes; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*
D005456:Fluorescent Dyes; D015850:Interleukin-6