Genetic Techniques

PCR - Polymerase Chain Reaction
1. Dna to be amplified, copies identical Sequence of DNA, Start martial could be DNA, RNA, archival speicmen DNA, cloned DNA, foresic samples
2. 1. Denaturation of Double strand of DNA
  1. DsDNA - Temp -93-95'C -30 sec
  2. 2. Annealing
    1. Temp - 40-70'C -30 sec
    2. 3.DNA Synthesis
      1. Temp-70-75'C - 1 minute
      2. DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template.
    3. This allows the primers to bind (anneal) to their complementary sequence in the template DNA
  3. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the strands to separate creating single stranded DNA.
3. Product size - Length of forward primer + Length of reverse primer + region to be ampilfied
  1. Think - DNA replication outside the body
4. There are five basic reagents, or ingredients, necessary for PCR: template DNA, PCR primers, nucleotides, Taq polymerase and PCR buffer.
  1. Template DNA
    1. starting DNA that is used
  2. PCR Primer
    1. is a short piece of DNA that identifies the region of DNA
  3. Nucleotides
    1. subunit of a DNA chain - A,T,C,G
  4. Taq Polymerase
    1. DNA polymerase from a bacterium called Thermus aquaticus
  5. PCR Buffer
    1. PCR buffer is a solution that optimizes conditions like salt concentration and pH to enable Taq polymerase to work efficiently
Why is PCR used?
1. Diagnosis and screening of genetic diseas
  1. Detection of slowly growing microoranisms
    1. HLA transplantation
      1. DNA finger prints
        Cloning/ Mapping
Polymorphism detection
1. DNA polymorphism - types of mutations and variable number of ministallite repeats (VNRS)
RT PCR - 1 - 1. the RT reaction and PCR amplification. RNA is first reverse transcribed into cDNA using a reverse transcriptase as described here, 2. the resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes.
1. DNA Copies for RNA targets RT PCR
  1. Transcription rates
    1. Cloning of rare gene
Genetic Engineering
1. Process that manipulates Genes - normally reproductive process
2. Usually involves isolation, manipulation and reintroduction of DNA into cells or model Organism
3. Recombinant DNA technology
  1. INSULIN
  2. Basic procedure: 1. DNA purified from cells or tissues 2. Restriction enzymes used to generate specific DNA fragments recognise and cut DNA at specific sequence 3. Fragments join to other DNA molecules which act as vectors (carrier molecules), vector + DNA fragment= recombinant DNA molecule 4. Recombinant DNA molecule transferred to host cell 5. Within host cell recombinant replicates producing dozens of identical copies 6. As host replicates, recombinant molecule passed to all progeny creating population of cells each carrying cloned DNA sequence 7. Cloned DNA can be recovered from host cells, purified and analysed or 8. Cloned DNA in host cells is transcribed, its mRNA translated, gene product isolated and used or sold.
4. CLoning
  1. Reproductive
    1. Is the creation of an exact genetic replica of a small segment of DNA, a cell or a whole organism
      1. Dolly the sheep
  2. Therapeutic cloning
    1. Stem cells
5. Gene therapy
  1. Used for correcting defective gene, normal gene is inserted to replace abnormal gene
    1. 1. Gene Augmentation Therapy – The introduction of DNA into a cell with a view to augmenting gene function, e.g. recessivediseases. 2. Targeted Modulation of gene expression – Methods for turning off genes using for example ribozymes and anti- sense oligonucleotides, e.g., infectious disease and cancers
      1. Difficulties - only good for LOF, targeting gene needed for treatment, correct level of gene expression, altering pheontype
        Shift to cancer and HIV - genetic disease
Genetic Counselling
1. A child is born with several abnormalities There is a history in the family of a medical condition A woman has a history of several miscarriages Woman aged over 35 yrs and pregnant Anyone who has uncertainties about genetic risks
  1. Genetic testing
    1. Pre-pregnancy- blood test
    2. In pregnancy - CVS -11-12 weeks or Amniocentesis - 15-19 weeks
    3. After pregnancy - Guthrie test, blood test

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Genetic Techniques

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	Created by Clarissa Mackay over 8 years ago