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Polymerase C hain R eaction (PCR) New Tool in Molecular Diagnostic

Dr. M. Sasvári : Recombinant DNA Technology, I. Polymerase C hain R eaction (PCR) New Tool in Molecular Diagnostic. Http://www.biokemia.sote.hu. PCR. (2 x ). 10 2 -10 3 bp PCR product. Amplification of a desired dsDNA fragment. Total human genome. 2 x 3x10 9 bp 2 x 23 chromosomes.

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Polymerase C hain R eaction (PCR) New Tool in Molecular Diagnostic

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  1. Dr. M. Sasvári : Recombinant DNA Technology, I. Polymerase Chain Reaction (PCR) New Tool in Molecular Diagnostic Http://www.biokemia.sote.hu

  2. PCR (2 x ) 102-103 bp PCR product Amplification of a desired dsDNA fragment Total human genome 2 x 3x109 bp 2 x 23 chromosomes dsDNA fragment

  3. DNA DNA : „oligo” or primer, 16-20 bp synthetic ssDNA Specificity of PCR amplification At least two select the two ends of the PCR product ?

  4. DNA DNA 2. cycle PCR amplification is exponential (duplicating in every cycle) 1 dsDNA  106 fragments / 25 cycles (approx.. 3 hours)

  5. Metal thermoblock block (fast changes in temperature) The PCR reaction 20-50 ml of reaction mixture • template DNA • primers • dNTP + Mg2+ • Taq DNA polymerize (Thermus aquaticus) • HEAT STABLE

  6. The thermocycle I. Denaturation (96oC) I. dsDNA  ssDNA III. II. T t t II. Annealing40-60oC) primers bound to the complementary sequences III.Polymerization (72oC) DNA replication with the heat stable polymerase

  7. Screening for disease causing mutations Risk factors Polygenic inheritance The DNA sample Medical application of PCR Genetic canceling Monogenic disorders lethal/treatable diseases: prenatal diagnosis E.g. oncogenes invasive DNA sampling: blood (Guthrie spot) CVS (chorion villi sample) non-invasive sampling Buccal cells hair, nail

  8. Bacterial and viral sequences: not found in the human genome Examples: Medical application of PCRIdentification of a foreign genome Detection: specific primers - is there any PCR product? Pneumococcus (tubercolosis) - “sputum” HIV (?) Clamidomas

  9. Other applications DNA “fingerprinting” Paternity cases Criminology (hair, blood-spot, etc.) Immigration (relationship)

  10. agarose gel Big Ethidium bromide staining (intercalation) – UV 100 V + Small ladder Separation and visualization of PCR products

  11. 3. step: gel electrophoresis 1. step: PCR amplification : - patient’s number 2. 4. 1. 3. A T T A normal gene Mutant gene 2. step: Cleavage with Mst II. restriction endonuclease: + Q: Which patient is a carrier? Who will have SSD? Methods of mutation analysis PCR-RFLP e.g.:Sickle cell disease (SSD), hemoglobin b-chain Glu6Val6

  12. Gel electrophoresis - Primer construction : 2. 4. 1. 3. CFTR gene + Q:If this is a prenatal diagnosis (parents: 1 and 2), which sample would belong to a healthy embryo (3 or 4)? Detection of a deletion e.g.: Cystic fibrosis DF 508: 3 bp deletion (Phe minus) normal product mutant product

  13. X-linked inheritance ( DMD frequency 1: 3500 men) Deletion sites 1. 4. C A B 2. 3. Q: Which of the patients has DMD? Who has probably the most sever symptoms? Identification of larger deletions e.g.: DMD (Duchenne muscular dystrophy) The longest known gene (2 million bp) Very long protein product: Dystrophin (Mw 426 kD) Primer construction Primers are complementary to the deleted sequences

  14. Identification of mutations ASA: Allele Specific Amplification Principle of ASA: if the 3`end of the primer is not complementary to the template, no PCR product is formed Medical importance of prenatal diagnosis in case of 21-steroid hydroxylase deficiency (CYP21 gene) CYP 21 mutations: disturbance of steroid metabolism Underproduction of aldosterone  SALT WASTING Overproduction of androgenes  VIRILIZATION Prenatal diagnosis - fetal treatment in females

  15. ASA: Allele Specific Amplification DNAN ... ... PN: normal primer 3’ ... ... DNAN PMt: mutant primer 3’ DNAMt ... ... PN: normal primer 3’ DNAMt ... ... PMt: mutant primer 3’ STOP STOP

  16. Genotyping with ASA PMt PMt PN PN Internal control Allele specific product Patient 1 Patient 2 Patient 3 Q: Which of the patients are healthy/carrier/sick (homozygous for the mutation)? PMt PN

  17. Recombinant DNA technology Production of human recombinant proteins for pharmaceutical purposes

  18. Restriction endonuclease(RE) Plazmid vector recombinant vector Foreign DNA RE AmpR Ligation DNS ligase Principles of vector construction

  19. Expression vectors - insert contains only exons (cDNA) - bacterial promoter - bacterial ribosome binding sites Turning on and off the protein production Bacterial DNA lac operator lac promoter cDNA of Human protein Repressor protein +IPTG induction expression Expression of a Human gene in Bacteria

  20. 1. Vector construction operator lacZ (b gal) promoter Artificial “ insulin gene” AmpR Insulin A or B chain (insert) ori Insulin: The first licensed recombinant protein Amino acid sequence  base sequence

  21. bgal insulin 3. IPTG added  fusion protein is produced 4. Lysis, isolation and purification of the protein 5. CN Br treatment: Methionins are broken, insulin released 2. Transformation, selection of AmpR clones, cloning E. coli plasmid AmpR lac repressor Bacterial chromosome 6. A and B chain fold in vitro

  22. Bacterial production of Met-less hGH 1. vector construction hGH Bacterial signal sequence (extracellular) 1. Transformation selection growth 2. Secretion of hGH into the periplasmic space 3. Bacterial periplasmic protease: cuts the signal sequence 4. Met-less rec-hGH, purification AmpR ori

  23. Transgenic animals Transient expression Microinjection of the fertilized egg advantage: stabile integration random integration (ES cell transformation: homologue recombination) Embrionic Stem cells ‘Knockout’ and ‘knockin’ animals Expression of human gene in mammals Non-integrating viral vectors e.g. adenovirus advantage: no transformation disadvantage: temporal

  24. EuK cell Viral-vector-reporter gene construct nucleus Expression assay based on the reporter protein Reporter proteins • luciferase ATP  ADP + Pi + light • b-galactosidase Lactose analogue  blue product BLUE CELLS • CAT (chloramphenicol acetyl transferase) Transient expression: reporter genes

  25. Application of reporter genes e.g. testing trans -activating proteins (TF) 2 3 reporter 1 1: enhancer 2: promoter 3: gene of TF Vector B vector A EuK cell nucleus TF

  26. Transgenic animals 1982: The giant mouse Fertilized oocyte Suction pipette vacuum Microinjection of hGH cDNA into the male pronucleus

  27. Preparation of transgenic animals 1. Microinjection (many hundreds of eggs, micromanipulator) 2. Implantation of the egg into a pseudo-pregnant mouse 3. Check the pubs (PCR from the tail) 4. Mate with transgenic animal 5. Inbreeding - Cloning as a new alternative ?

  28. Production of secretory recombinant proteins Microinjection of mouse with t-PA gene + lactalbumin signal sequence and a proper regulatory unit secretion of t-PA into the milk: 0.1 mg t-PA/ml milk of the mouse Future: transgenic (cow?), goat, sheep Genetically modified crops Molecular Farming

  29. Vector: • a circular dsDNA molecule • suitable for inserting a DNA fragment • replicates after introducing into a host cell Types of vectors: • plasmid (extrachromosomal DNA) • phage (bacterial host) • virus (plant, animal, human hosts) Genomic library A series of recombinant phages/cosmids containing different DNA fragments of the human genome Cosmid: • artificial plasmid • a combination of phage COS sequence + a plasmid • similar to plasmids: antibiotic resistance gene • replication origo • can take a large insert (40 kb) • similar to phage: infects E. coli after packaging in vitro Basic terms (see Lehninger: Recombinant DNA technology) Maximal size of DNA that can be cloned in vectors plasmid 20 kb - phage 25 kb cosmid 45 kb YAC 1000 kb (yeast artificial chromosome) Restriction enzymes recognition site / fragment size 4 bp 256 bp 6 bp 4000 bp 8 bp 64000 bp

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