Transcript In Search of the Sickle Cell Gene

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
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Facilitates transfer of DNA onto nylon membrane
Add labeled probes which attach to target DNA
Analyze
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