Rapid sizing of polymorphic microsatellite markers by capillary array electrophoresis.
Genetic mapping and DNA sequencing projects could potentially be completed more rapidly by using capillary array electrophoresis (CAE) systems running 48-96 capillaries simultaneously. Currently, multiplex polymerase chain reaction (PCR) and multicolor fluorescent dye-labeling strategies are used to generate DNA profiles containing 18-24 genotypes per sample. By using 4-color fluorescence detection and these multiplex PCR strategies, a CAE system has the capacity to generate up to 5.5 million genotypes per year. CAE offers extremely fast, high-resolution separation of DNA and more automated sample processing than conventional systems because the labor-intensive slab-gel pouring and sample-loading steps are eliminated. We used a prototype CAE system in an ongoing linkage analysis study of inherited deafness in Mediterranean families. CA-repeat markers linked to deafness susceptibility genes on chromosomes 7, 11 and 13 were analyzed and DNA profiles generated which contain 6 markers per color. Fragment sizes of over 28,000 short tandem repeat alleles and 3200 CA-repeat alleles have been determined by CAE. An average sizing precision of +/- 0.12 base pairs (bp) for fragments up to 350 bp was realized in 1-h runs. In addition, a versatile non-denaturing matrix was used to separate DNA sizing standards, restriction digests, and multiplex PCR samples. Application of this matrix to Duchenne muscular dystrophy exon deletion screening is also described. These CAE approaches should facilitate rapid genotyping of microsatellite markers and subsequent identification of disease-causing mutations.
Mansfield
E S
ES
Molecular Dynamics, Sunnyvale, CA, USA.
Vainer
M
M
Harris
D W
DW
Gasparini
P
P
Estivill
X
X
Surrey
S
S
Fortina
P
P
D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D008040:Genetic Linkage; D005838:Genotype; D018895:Microsatellite Repeats / Q000235:genetics*; D010375:Pedigree; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers