pcr & dystrophin gene

pcr & dystrophin gene

• 1.
  PCR & Dystrophin gene
• 2.
  Polymerase Chain Reaction (PCR) WHATIS PCR?  Was originally developed in 1983 by the American biochemist Kary Mullis.  PCR is used in molecular biology to make many copies of (amplify) small sections of DNA or a gene  To generate thousands to millions of copies of a particular section of DNA from a very small amount of DNA.  USES:  cloning, genetic engineering, sequencing (for detecting the presence or absence of a gene to help identify pathogens during infection)
• 3.
  COMPONENTS FUNCTION DNA templatethe sample DNA that contains the target sequence DNA polymerase enzyme that synthesizes new strands of DNA complementary to the target sequence. (Taq DNA polymerase (from Thermis aquaticus), Pfu DNA polymerase (from Pyrococcus furiosus)) Primers short pieces of single-stranded DNA that are complementary to the target sequence that initiate the PCR reaction Nucleotides (dNTPs or deoxynucleotide triphosphates) single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands. Buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase HOW DOES A PCR WORKS? The PCR reaction requires the following components:
• 4.
  STAGES Denaturation Annealing Elongation
• 5.
  1. Denature DNA TheDNA is heated to 95° C. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the strands to separate creating single stranded DNA. 2. Primer Annealing The mixture is cooled to anywhere from 45-72° C. This allows the primers to bind (anneal) to their complementary sequence in the template DNA. 3. Extension The reaction is then heated to 72° C, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template. EXPLAINATION **These three stages are repeated 20-40 times, doubling the number of DNA copies each time. After PCR has been completed, a method called ELECTROPHORESIS can be used to check the quantity and size of the DNA fragments produced.
• 6.
  Dystrophin Gene  Thelargest known human gene (vulnerable to mutation)  Provides instructions for making a protein called Dystrophin.  LOCATION: (i) Short (p) arm of the X chromosome at position 21.2. (Xp21.2) (ii) Primarily in muscles used for movement (skeletal muscles) and in heart (cardiac) muscle. Small amounts of dystrophin are present in nerve cells in the brain.
• 7.
  Dystrophin Protein  Dystrophinis a protein located between the sarcolemma and the outermost layer of myofilaments in the muscle fiber (myofiber).  It is a cohesive protein, linking actin filaments to another support protein that resides on the inside surface of each muscle fiber’s plasma membrane (sarcolemma).  This support protein on the inside surface of the sarcolemma in turn links to two other consecutive proteins for a total of three linking proteins. The final linking protein is attached to the fibrous endomysium of the entire muscle fiber Forms part of a complex known as the dystrophin- glycoprotein complex (DGC). Dystrophin forms a link between the outside the inside of the muscle membrane. OUTSIDE INSIDE
• 8.
  Role of Dystrophinprotein • In skeletal and cardiac muscles, dystrophin supports and strengthen muscle fibers and protect them from injury as muscles contract and relax. The dystrophin complex acts as an anchor, connecting each muscle cell's structural framework (cytoskeleton) with the lattice of proteins and other molecules outside the cell (extracellular matrix). The dystrophin complex may also play a role in cell signaling by interacting with proteins that send and receive chemical signals
• 9.
  Dystrophin gene Mutation (1)Deletion, (2)Frameshift mutation, (3) Point mutation • Loss of sarcolemma integrity • Excessive influx of Ca2+ • Increase release of soluble muscle enzymes (Creatinine Kinase) into serum
• 11.
  Indication of PCR diagnosis•Multiplex polymerase chain reaction (PCR) is used to screen for exon deletions within the dystrophin gene • Multiplex PCR is a technique in which several PCR- products are amplified in one PCR-reaction. • DMD is an X-linked disease, i.e. only one X- chromosome is present. Consequently, deletion of an exon can be determined by the failure to amplify that exon by PCR.