Detection of allelic variants of the canine IGHA gene by fluorescence resonance energy transfer melting temperature examination.
The fluorescence resonance energy transfer (FRET) dual hybridisation probe system has been used for the detection of the accumulation of target DNA during real-time PCR and for the identification of nucleotide polymorphisms through examination of melt curves. This system involves the use of two oligonucleotide probes which are located close to each other and are complementary to an internal segment of a target DNA of interest. Four allelic variants of the gene encoding the hinge region of the immunoglobulin A (IgA) heavy chain (IGHA) have been so far identified in the dog and this variability is due to a combination of single nucleotide polymorphisms and insertion/deletion of nucleic acid motifs. An individual dog may be homozygous or heterozygous for these allelic variants. The purpose of this study was to develop a FRET-based dual probe melting temperature assay to identify the alleles present within an individual dog and to use this assay to determine the frequency of the four allelic variants in different breeds within the canine population. A single pair of oligonucleotide probes were designed that were able to discriminate between the four allelic variants in both homozygous and heterozygous individuals. The genotype of 96 DNA samples obtained from various purebreeds of dogs was determined using this FRET assay. The frequency of each allele differed between the breed groups. The results of this study indicate that it is possible to distinguish relatively complex gene polymorphisms using a single set of oligonucleotide probes. Furthermore, any future comparison of IGHA genotypes between normal and diseased dog populations must take into account the breed variation in allelic frequency.
Peters
I R
IR
School of Clinical Veterinary Science, University of Bristol, Langford House, Langford, Bristol BS40 5DU, UK. I.R.Peters@Bristol.ac.uk
Helps
C R
CR
Lait
P L
PL
Harris
C
C
Lee
A C
AC
Jones
C A
CA
Hall
E J
EJ
Day
M J
MJ
D000483:Alleles; D000818:Animals; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D014644:Genetic Variation; D005838:Genotype; D006358:Hot Temperature; D007070:Immunoglobulin A / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction
D007070:Immunoglobulin A; D007143:Immunoglobulin Heavy Chains; D015345:Oligonucleotide Probes