MLAB 2321 Molecular Diagnostics For Clinical Laboratory Science

Unit 11 Objectives - Gene Mutations
Textbook Chapter 9

  1. Give a brief description of the techniques used by filling in this Detection of Gene Mutations chart . (This chart can be referred to during Exam 3)
  2. Explain why knowledge of the effects of different types of mutations is important when interpreting the results of mutation analyses.
  3. State why it is more difficult and challenging to detect somatic mutations.
  4. Briefly describe the three sequence detection methods: hybridization based methods, sequence (polymerization) based methods and enzymatic or chemical cleavage methods.
  5. State the four basic constituents of a molecular genetic test.
  6. List the three variables taken into consideration in selecting a testing method.
  7. State the basic consideration for using a method with high sensitivity and specificity used for screening versus a method used to define a specific target.
  8. Given a mutation, use your textbook to write the mutation using the correct gene mutation nomenclature.
  9. Define or describe the terms listed in the "Unit 11 Glossary of Terms".

(a) Several methods to detect specific nucleotide changes (polymorphisms) exist. One method relies on hybridization of oligonucleotides of known sequences to target DNA. The target DNA is generally obtained using the polymerase chain reaction and specific primers. Allele-specific oligonucleotides are then used to detect single base changes in the DNA samples. Typically, target DNA is immobilized on a solid support and denatured. Labeled (radioactive or fluorescent) oligonucleotides are then allowed to anneal. Complementary sequences bind while noncomplementary sequences do not. Sequences that match the oligonucleotide are detected by fluorescence or when the oligonucleotide is radiolabeled by exposure to X-ray film. (b) Another means of rapid screening for DNA variations relies on detecting conformational changes in secondary structure caused by the nucleotide sequence alteration. The change in structure can be detected in a number of ways including denaturing gradient electrophoresis and denaturing gradient high-performance liquid chromatography. SSCP, single-stranded conformational polymorphism. (c) Base mismatch methods begin with creating heteroduplexes between wild-type or normal DNA and target DNA. Heteroduplexes with mismatches are detected by enzymatic or chemical cleavage, with the cleavage products resolved by electrophoresis. (d) DNA sequencing can also be used to detect polymorphisms but is the most labor intensive. The method involves synthesis of DNA using DNA polymerase. Dideoxynucleotides are included in the synthesis mix to randomly terminate synthesis at each nucleotide in the sequence. Generally, each dideoxy nucleotide is labeled with a flourescent tag. Terminated strands are separated by denaturing gel or capillary electrophoresis and are detected using fluorescence.

Reference: Iannuzzi et al. Respiratory Research 2002 3:15 doi:10.1186/rr164

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