In Search of the Sickle Cell Gene

Terry Kotrla, MS, MT(ASCP)BBSpring 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• http://tinyurl.com/26kzopy