In Search of the Sickle Cell Gene Terry Kotrla, MS, MT(ASCP)BB Spring 2010

Introduction Single nucleotide change in DNA can affect health and cause disease Large number of genetic diseases have been identified. Mutations in oncogenes and tumor suppressor genes associated with lung, colon and breast cancer (BRCA I and II).

Genetics Predicts traits inherited by offspring Based on inheritance of two alleles, two forms of the gene, one from each parent Can be dominant, only 1 gene necessary for expression Or recessive, 2 identical genes necessary for expression. Dominant allele can mask recessive gene, trait Page 5 Figure I

Sickle Cell Gene Sickle cell anemia caused by defective hemoglobin. Autosomal recessive genetic disorder. – Normal hemoglobin gene A – Sickle hemoglobin gene S In normal hemoglobin A, glutamic acid is on the 6th position of the beta chain. In sickle-cell disease glutamic acid is replaced by valine leading to the formation of sickle cells. Single base mutation is A to T

Identifying the Gene Electrophoretic comparison of Hb S and Hb A – Slightly basic pH Hb S more positive than Hb A – Hb S will travel more slowly toward the anode. – Able to separate out Fetal DNA can be obtained from cells obtained by amniocentesis. Normal CCT-GAG-GAG Point mutation changes A to G so sequence is CCT-GTG-GAG

Southern Blot Used for analysis of DNA Perform electrophoresis to separate Treat with HCl and NaOH. HCL nicks double stranded DNA causing apurinic sites NaOH disrupts intrastrand hydrogen bonds between base pairs Results in formation of small fragments from large DNA fragments Facilitates transfer of DNA onto nylon membrane Add labeled probes which attach to target DNA Analyze