Screening strategies for a highly polymorphic gene
Standard
Screening strategies for a highly polymorphic gene : DHPLC analysis of the Fanconi anemia group A gene. / Rischewski, J; Schneppenheim, R.
In: J BIOCHEM BIOPH METH, Vol. 47, No. 1-2, 30.01.2001, p. 53-64.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
Harvard
APA
Vancouver
Bibtex
}
RIS
TY - JOUR
T1 - Screening strategies for a highly polymorphic gene
T2 - DHPLC analysis of the Fanconi anemia group A gene
AU - Rischewski, J
AU - Schneppenheim, R
PY - 2001/1/30
Y1 - 2001/1/30
N2 - INTRODUCTION: Patients with Fanconi anemia (Fanc) are at risk of developing leukemia. Mutations of the group A gene (FancA) are most common. A multitude of polymorphisms and mutations within the 43 exons of the gene are described. To examine the role of heterozygosity as a risk factor for malignancies, a partially automatized screening method to identify aberrations was needed. We report on our experience with DHPLC (WAVE (Transgenomic)).METHODS: PCR amplification of all 43 exons from one individual was performed on one microtiter plate on a gradient thermocycler. DHPLC analysis conditions were established via melting curves, prediction software, and test runs with aberrant samples. PCR products were analyzed twice: native, and after adding a WT-PCR product. Retention patterns were compared with previously identified polymorphic PCR products or mutants.RESULTS AND DISCUSSION: We have defined the mutation screening conditions for all 43 exons of FancA using DHPLC. So far, 40 different sequence variations have been detected in more than 100 individuals. The native analysis identifies heterozygous individuals, and the second run detects homozygous aberrations. Retention patterns are specific for the underlying sequence aberration, thus reducing sequencing demand and costs. DHPLC is a valuable tool for reproducible recognition of known sequence aberrations and screening for unknown mutations in the highly polymorphic FancA gene.
AB - INTRODUCTION: Patients with Fanconi anemia (Fanc) are at risk of developing leukemia. Mutations of the group A gene (FancA) are most common. A multitude of polymorphisms and mutations within the 43 exons of the gene are described. To examine the role of heterozygosity as a risk factor for malignancies, a partially automatized screening method to identify aberrations was needed. We report on our experience with DHPLC (WAVE (Transgenomic)).METHODS: PCR amplification of all 43 exons from one individual was performed on one microtiter plate on a gradient thermocycler. DHPLC analysis conditions were established via melting curves, prediction software, and test runs with aberrant samples. PCR products were analyzed twice: native, and after adding a WT-PCR product. Retention patterns were compared with previously identified polymorphic PCR products or mutants.RESULTS AND DISCUSSION: We have defined the mutation screening conditions for all 43 exons of FancA using DHPLC. So far, 40 different sequence variations have been detected in more than 100 individuals. The native analysis identifies heterozygous individuals, and the second run detects homozygous aberrations. Retention patterns are specific for the underlying sequence aberration, thus reducing sequencing demand and costs. DHPLC is a valuable tool for reproducible recognition of known sequence aberrations and screening for unknown mutations in the highly polymorphic FancA gene.
KW - Base Sequence
KW - Child
KW - Chromatography, High Pressure Liquid
KW - DNA Mutational Analysis
KW - DNA Primers
KW - Exons
KW - Fanconi Anemia
KW - Genetic Testing
KW - Genetic Variation
KW - Hematologic Neoplasms
KW - Heterozygote
KW - Homozygote
KW - Humans
KW - Leukemia, Myeloid, Acute
KW - Nucleic Acid Denaturation
KW - Polymerase Chain Reaction
KW - Polymorphism, Genetic
KW - Polymorphism, Single Nucleotide
KW - Reproducibility of Results
KW - Risk Factors
M3 - SCORING: Journal article
C2 - 11179761
VL - 47
SP - 53
EP - 64
JO - J BIOCHEM BIOPH METH
JF - J BIOCHEM BIOPH METH
SN - 0165-022X
IS - 1-2
ER -