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PCR Genomic technologies

PCR Genomic technologies. PCR. "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat.".

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PCR Genomic technologies

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  1. PCR • Genomic technologies

  2. PCR "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat." Kary Mullis. Nobel prize in chemistry, 1993

  3. Studies on Polynucleotides: Repair Replication of Short Synthetic DNA’s as catalyzed by DNA Polymerases. K. KLEPPE, E. OHTSUKA, R. KLEPPE, I. MOLINEUX and H.G.KHORANA J. Mol Biol (1971) PAIRS OF PRIMERS AND REPEATED CYCLING MAY LEAD TO EXPONENTIAL AMPLIFICATION OF THE TEMPLATE.

  4. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Randall K. Saiki, Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, Norman Arnheim Science, 1985 Each Cycle (20 carried out) Denaturation step: 95oC Annealing step: 30oC Extension/elongation step: 30oC BUT……

  5. FINDING A THERMOSTABLE POLYMERASE! THERMUS AQUATICUS Taq Polymerase (75-80oC) 1:9000 error rate Pfu DNA Polymerase (75-80oC) 1:1,300,000 error rate Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase. R.K. SAiK, D.H. GELFAND, S. STOFFEL, S. J. SCHARF, R. HIGUCHI, G.T. HORN, K.B. MULLIS, H.A. ERLICH. Science. 1988

  6. BASIC PCR PROTOCOL Denaturation step: 94–98°C for 20–30 seconds. Annealing step: 50–65°C for 20–40 seconds 3–5°C below the Tm of the primers used. Extension/elongation step: 75–80°C,(1000 bases per minute). Final elongation: 70–74°C for 5–15 minutes after the last PCR cycle. Final hold: 4–15°C for an indefinite time.

  7. CONSIDERATIONS FOR PRIMER DESIGN 1) Primer Length A, C, T, G so 1:4 chance of each nucleotide at each position For a 12mer then we have 0.2512 chance of each nucleotide at each Position = 1:16.8x106. 3.3x109 /16.8x106 = 196! For an 17mer then we have 0.2518 chance of each nucleotide at each Position = 1:17.18x109. 3.3x109 / 1:17.18x109 = 0.2! 18-30mers are therefore typical

  8. CONSIDERATIONS FOR PRIMER DESIGN 2) Annealing temperature AATTCGATTTCGGTTATTACT (54oC) A T G C T A C G CCGGACATGACCGATTCTACC (66oC) 52-58oC. 40-60% GC content. 3) GC clamp TTACCTATCACGACATCG |||||||||||||||||| ………..AATGGATAGTGCTGTAGCTAATGTGACTCGGTAATGTAGG……………………….

  9. CONSIDERATIONS FOR PRIMER DESIGN 4) Secondary structure and primer/dimer Vector NTI Primer3 BioTools

  10. CONDITIONS FOR PCR REACTIONS • Template DNA (0.5 - 50 ng) • < 0.1 ng plasmid DNA, 50 ng to 1 μg gDNA for single copy genes • Oligonucleotide primers (0.1 – 2.0 μM) • dNTP’s (20 –250 μM) • Thermostable DNA pol (0.5 – 2.5 U/rxn) • MgCl2 (1 – 5 mM) affects primer annealing and Taq activity • Buffer (usually supplied as 10X) • Working concentrations • KCL (10 – 50 mM) • Tris-HCl (10 mM, pH 8.3) • NaCl2 (sometimes)

  11. USES FOR PCR

  12. 1) Analysis of genomic mutations CCR5 Sickle cell anemia (b-globin)

  13. 2) Forensic Applications Restrictive sources of DNA Variable number tandem repeats VNTRs

  14. 3) Gene Expression Analysis D6 Actin 0 8 16 24 0 8 16 24 TPA Acetone

  15. Loading Controls Commonly used controls are: Glyceraldehyde-3-phosphate dehydrogenase Beta actin MHC I (major histocompatibility complex I) Cyclophilin Tata Binding Protein 28S or 18S rRNAs (ribosomal RNAs) D6 D6 Actin 0 0 8 8 16 16 24 24 0 0 8 8 16 16 24 24 TPA TPA Acetone Acetone

  16. 10 1 0.1 0.01 0.001 0.0001 il-4 il-6 pf4 il-1b itgm Fpr1 4) Real-Time Quantitative PCR CT Absolute: compared to a standard Relative: compared to a housekeeping gene * ** ) * Ct D - 2 ** *** * Log10 ( D6 Actin Sybr-Green 0 8 16 24 0 8 16 24 TPA Acetone

  17. Bcrablfusion protein Strong kinase activity 5) Minimal Residual Disease Bcr (chr22) Abl (chr9) Weak kinase activity MOLECULAR REMISSION!

  18. Single Cell PCR Single cell gene, and gene expression, analysis. Biological noise and complexity!

  19. PCR • Genomic technologies

  20. THE HUMAN GENOME MAPPING PROJECT SEEKS TO READ THE FULL SEQUENCE OF THE HUMAN GENOME

  21. Genome Sequencing Sanger Sequencing Dye Terminator sequencing

  22. Genome Sequencing This requires advanced computing, statistics and mathematics. Bioinformatics. Massively parallel sequencing

  23. Information from all these genomes help inform our understanding of the Human genome. Differentiation Cell death (apoptosis) Cell cycle Other species genomes are also being sequenced!

  24. transcribe RNA translate Protein THE GENOME IS NOW DEFINED! 3 Billion bases in each cell Our genes (23,000) only occupy 3% of the genome!

  25. ALL CELLS HAVE THE SAME DNA!!! So, why do we have so many different cell types Different cells transcribe different sets of genes A B C D E F G H I J Skin cell Blood cell Brain cell

  26. ALL CELLS HAVE THE SAME DNA!!! So, why do we have so many different cell types Different cells transcribe different sets of genes A B C D E F G H I J Skin cell Blood cell Brain cell SAME GENOME, DIFFERENT TRANSCRIPTOME

  27. We can now start to define an expression fingerprint for each cell type – a transcriptional profile

  28. Transcriptional profiling by micro-array analysis This technology has given rise to the field of ‘Transcriptomics’.

  29. Transcriptional profiling by micro-array analysis Normal Cell Cancer cell 10 healthy control samples 10 cancer patient samples 40,000 gene variants 140 probes per gene Key gene differences 112,000,000 data points!

  30. Bioinformatics As an interdisciplinary field, bioinformatics combines computer science, statistics, mathematics and engineering to study biological data and processes. Bioinformatics analysis is unbiased!

  31. Transcriptional profiling by micro-array analysis In this way we can define the transcriptome of a cell!

  32. THE GENOME: INVARIANT! THE TRANSCRIPTOME: VARIES IN DIFFERENT CELL TYPES

  33. transcribe RNA translate Protein THE GENOME IS NOW DEFINED! 3 Billion bases in each cell Our genes (23,000) only occupy 3% of the genome! Exons Introns Promoter Micro-RNAs

  34. miR146a ugagaacugaauuccauggguu (22mer)

  35. 1% protein coding 3% Genes 90% of SNPs outside protein coding regions

  36. transcribe RNA translate Protein THE GENOME IS NOW DEFINED! 3 Billion bases in each cell 80% of the genome is functional! Probably nearer 20%! Exons Introns Promoter Micro-RNAs

  37. The era of ‘deep-sequencing’! 1) Pathogen sequences 2) Protection of threatened species 3) Pre-historic animal genomes

  38. Using genome data to recreate ‘life’

  39. The era of ‘deep-sequencing’! 1) Pathogen sequences 2) Protection of threatened species 3) Pre-historic animal genomes 4) Rapid identification of disease causing genes

  40. Personalised Medicine The right drug for the right patient at the right time. EGFR mutations in NSCLC and gefitinib. EGFR2 (HER2) mutations in breast cancer and Trastuzumab.

  41. Personalised Medicine 1) Genomic sequencing 2) Single Nucleotide Polymorphism (SNP) analysis ATTGGCATGTGACC ATTGGCAAGTGACC 3) Transcriptomic analysis Gene expression that is diagnostic and predictive of response to therapy.

  42. PCR • Genomic technologies

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