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MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

MLAB 2401: Clinical Chemistry Keri Brophy-Martinez. Nucleic Acids and Molecular Testing. Nucleic Acid Probes / Molecular Testing. Assays for detection and quantitation of DNA and RNA Purposes Evaluation of genetically based diseases Detection of infectious organisms

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MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

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  1. MLAB 2401: Clinical ChemistryKeri Brophy-Martinez Nucleic Acids and Molecular Testing

  2. Nucleic Acid Probes / Molecular Testing • Assays for detection and quantitation of DNA and RNA • Purposes • Evaluation of genetically based diseases • Detection of infectious organisms • Determination of differences between individuals for forensic or transplantation purposes • Evaluation for altered cell growth regulation

  3. Nucleic Acid Probes / Molecular Testing • Hybridization Techniques • Nucleic acid probe • A short strand of DNA or RNA • Contains a well known sequence • Can be fragments of genomic nucleic acids or synthetic • Some are molecularly cloned in bacterial host

  4. Polymerase chain reaction (PCR) • Process to enzymatically synthesize millions of identical copies of a target DNA - increasing sensitivity • Three steps: denaturing, annealing, and extension. • Tissue, etc is digested with various enzymes to extract target DNA • Target DNA also called the target sequence

  5. Polymerase chain reaction (PCR) • Heat target DNA to denature and separate strands • Denaturing step • Two PCR primers are added to initiate the DNA synthesis • These are short pieces of single-stranded DNA that match the sequences at either end of the target DNA – serve somewhat like handles • Annealing (like pasting)

  6. Polymerase chain reaction (PCR) • An enzyme / DNA polymerase is needed to start and maintain the sequencing. • The enzyme must be able to withstand high temperatures, since the reaction occurs at @90 degrees C. • Taq polymerase (from Thermus aquaticus a heat resistant bacteria found in hot springs.) • Extension

  7. Polymerase chain reaction (PCR) • Addition of nucleotides used by the polymerase enzyme to make new DNA • Temperature lowered to @ 55 degrees C to facilitate this process. • Each time the process is repeated, more DNA copies are made. • The process with its repeated heating and cooling is best done in a PCR machine (aka thermocycler) which can increase and decrease the chamber temperature quickly.

  8. PCR • Limitations: expense, equipment, possibility for contamination, non-specific annealing, etc

  9. Polymerase chain reaction (PCR) • http://www.paternidad.com/science-technology/images/pcr.gif

  10. Nucleic Acid Probes / Molecular Testing • Nucleic Acid Probe Applications • detection of infectious organisms • detection of gene rearrangements • chromosomal translocation or breakage • changes in oncogenes or tumor suppressor factors • prenatal dx of inherited disease • identity or non-identity • donor selection

  11. Nucleic Acid Probes / Molecular Testing • Nucleic Acid Probe Applications • *** classic method for DNA analysis = Southern Blot

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