Lecture 13 Tools for Molecular Diagnosis

Lecture 13 Tools for Molecular Diagnosis - Define molecular...

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Define molecular cloning of a DNA segment Explain how DNA-DNA and DNA- RNA hybridization allows identification and isolation of specific fragments of DNA Describe the polymerase chain reaction (PCR) and its use in DNA fingerprinting Explain how fluorescent in situ hybridization (FISH) aids in the diagnosis of chromosomal abnormalities Describe how restriction enzyme length polymorphism (RFLP) experiments provide “DNA fingerprints” that can be used for several purposes Explain how the use of microarrays allows an analysis of the expression level of every gene in the genome Describe how the use of microarrays can provide information on the variant of cytochrome P450 carried by a patient, or tailoring during treatments of an individual to avoid side-effects (modified LO) Describe the advantages and disadvantages of widespread access to a patient’s genomic DNA sequence Vocab: Restriction enzymes, DNA cloning, northern and southern blots, polymerase chain reaction (PCR), fluorescent in situ hybridization, Restriction fragment length polymorphism (RFLP), single nucleotide polymorphisms(SNPS), cytochrome P450, microarrays, pharmacogenetics Outline: - The tools - The applications - The prospects - Molecular tools and techniques that we will discuss o Restriction enzymes o Gel electrophoresis o Nucleic acid hybridization o Northern blotting o RFLPs (restriction fragment length polymorphisms/SNPs) o Sanger) Dideoxy DNA sequencing o PCR (polymerase chain reaction) o Microarray analyses - Variations in sequences – DNA polymorphisms o o Frequent: 1/1000 bp for SNPs For micro- and minisatellites single changes vs multiple changes o Functional polymorphisms (polymorphisms may be within a gene and affect its function o Molecular polymorphisms – polymorphisms may be close by and serve as a marker (sickle cell anemia has a mutation in a flanking region) Motivations for identifying – DISEASE RELATED - Restriction Enzymes (enzyme produced by bacteria) o o Recognize and cleave specific DNA sequences (recognition and sequences o Inverted repeats (palindromic sequences) o Leaves compatible, sticky ends o Also: sticky ends vs blunt ends/endonucleases and exonucleaseas Define molecular cloning of a DNA segment Explain how DNA-DNA and DNA-RNA hybridization allows identification and isolation of specific fragments of DNA Describe the polymerase chain reaction (PCR) and its use in DNA fingerprinting Explain how fluorescent in situ hybridization (FISH) aids in the diagnosis of chromosomal abnormalities Describe how restriction enzyme length polymorphism (RFLP) experiments provide “DNA fingerprints” that can be used for several purposes Explain how the use of microarrays allows an analysis of the expression level of every gene in the genome Describe how the use of microarrays can provide information on the variant of cytochrome P450 carried by a patient, or tailoring during treatments of an individual to avoid side-effects (modified LO) Describe the advantages and disadvantages of widespread access to a patient’s
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