Презентация на тему "PCR and sequence"

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  Hybridization (DNA-DNA or DNA-RNA)

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  HYBRIDIZATION? –Yes, it is about this familiar picture

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  We can denaturate and renaturate DNA by heating/cooling

  http://www.biology.arizona.edu/molecular_bio/problem_sets/m/graphics/05ta.gif

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  As DNA is heated, it reaches a temperature where the strands separate (DNA melts).

  medlib.med.utah.edu/block2/ biochem/ ssDNA H-bonds between basepairs are broken and the strand unwind. Unstacked bases (random orientation) absorb more light than neatly stacked (oriented) base-pairs Melting curve. The temperature at which DNA is half unfolded Tm (meltingtemp) Tm is a measure of the stability of dsDNAunder a given set of conditions Hypochromic Shift

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  Tm of DNA is affected by:

  1. Base Composition: higher the GC content, the higher the Tm. 2. Ionic Strength : as the ionic strength increases, so does Tm. Double helical DNA is stabilized by cations. Divalent cations (Mg2+) are more effective than monovalent cations (NA+ or K+). 3. Organic Solvents – formamide for instance lowers the Tm by weakening the hydrophobic interactions.

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  medlib.med.utah.edu/block2/ biochem/ DNA more STABLE in high-salt conditions. DNA more STABLE When contains many GC

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  RNA can bind DNA (U is equivalent of T in hybridization)

  http://www.biology.arizona.edu/molecular_bio/problem_sets/m/graphics/05ta.gif

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  Hybridization could be less than perfect

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  medlib.med.utah.edu/block2/ biochem/ COMPLEX (DYNAMIC) PICTURE IN SOLUTION

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  http://amiga1.med.miami.edu/Medical/PDF-Files/Lecture10.pdf 42 C is more stringent condition that 35 C (hybridization is more specific)

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  So, hybridization is a most obvious phenomenon to use for specific DNA detection

  Specific probe self-anneals to target DNA Only problem – DNA is invisible How to visualize DNA? Radioactively Fluorescently

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  Polymerase labeling of DNA

  Gamma-33-ATP Alpha-32-ATP

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  Labeling by NICK TRANSLATION DNAse I Polymerase I (exonuclease activity) Polymerase I (polymerase activity) Will work without DNAse, as there are always nicks in DNA

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  T4 polynucleotide kinase labeling

  Gamma-33-ATP olig dNTP Used for oligonucleotide labeling

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  Professor Sir Ed Southern, Whitley Professor of Biochemistry at the University of Oxford

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  Allison, Fundamental Molecular Biology

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  “Real” Southern blot (DNA-DNA blot)

  www.mun.ca/biology/scarr/ 3250_Southern_Blot_analysis.htm STAINED by Et Br VIZUALIZED by P32

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  http://www.bseinquiry.gov.uk/report/volume2/fig1_8.htm Western Blot

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  Southern northern western What different types of information can be provided by each different blot type?

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  Colony hybridization assay for the identificationof bacterial colonies carrying a particular DNA clone

  http://amiga1.med.miami.edu/Medical/PDF-Files/Lecture10.pdf

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  http://biol1.bio.nagoya-u.ac.jp:8000/MatsudaM01fig2.jpg Same for arrayed clones

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  Design of degenerated synthetic hybridization probes (for already known proteins only)

  http://amiga1.med.miami.edu/Medical/PDF-Files/Lecture10.pdf

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  The degeneracy of a primer is the number of unique sequences it corresponds to (5 in one of the examples below).

  can be used when some of the related genomic sequences are unknown, or known only in a related species. Up to 1010 degeneracy is tolerated

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  Fluorescent probe hybridization

  A DNA probe, covalently bound to biotin, is hybridized to a denatured chromosome preparation. An avidin-bound fluorescent label (FITC) is layered on top of the cells, and the avidin-FITC binds the biotin. The signal is amplified further by layering rabbit anti-avidin antibody (which binds the avidin-FITC), and then layering FITC-labeled anti-rabbit antibody on top. Fluorescence will be detected only where the DNA probe has hybridized to the chromosome. http://www.childsdoc.org/spring2000/missinggenes.asp

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  How streptavidin/biotin binding is working?

  Largest free energies of association of yet observed for noncovalent binding of a protein and small ligand in aqueous solution (K_assoc = 10**14). Complex is extremely stable. Streptavidin is a protein. 1 mole of SA binds 4 moles Bio BIOTIN is a vitamin B (small thing) Biotin could be added to nucleotide and incorporated into the probe (67 kD protein from Streptococcus avidinii) Avidin could be conjugated with fluorophore

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  IN SITU HYBRIDIZATIONis an imaging method to visualize mRNA expression in tissues and cells. 

  Encephalin gene expression in the mouse brain www.omrf.org/OMRF/ Core/InSitu.asp The HuC transcript is expressed specifically in the nervous system of this E18 mouse

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  FISH labeling of the centromeric highly repeated DNA www.infobiogen.fr/.../ Metaphase FISH analysis using the BAC probe RP11-104M2 labeled with FITC (green) hybridized to a normal metaphase cell confirms the chromosomal localization of the probe (gene) to 4q28.

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  Cy3/Cy5 direct labelling of DNA (for microarrays)

  Cy5 -- RED

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  2. Polymerase chain reaction (PCR)

  http://www.bioteach.ubc.ca/MolecularBiology/PolymeraseChainReaction/PCR.gif

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  Polymerase Chain Reaction (PCR)

  ww2.mcgill.ca/biology/undergra/ c200a/f07-16.gif DNA melting Primer annealing DNA elongation Nobel Prize in Chemistry 1993, at age 48 Kary Mullis (invented PCR in 1983) PhD "The Cosmological Significance of Time Reversal," Biochemistry from U.C. Berkeley

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  Exponential nature of PCR amplification

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  www.biotechlab.nwu.edu/ pe/Sld022.htm

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  use OLIGONEW or PRIMER software Try for equal Tm for both primers

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  Avoid primer dimer formation

  Marginally problematic primer

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  Use Software to avoid of such problems

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  Typical PCR gel (Every PCR should by gel-verifyed)

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  Fidelity of PCR is often an issue www.biotechlab.nwu.edu/ pe/Sld022.htm mkM

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  www.biotechlab.nwu.edu/ pe/Sld022.htm Proof-reading activity enzymes are required for High Yield and High Fidelity are mutually exclusive

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  www.biotechlab.nwu.edu/ pe/Sld022.htm

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  If complete copies is amplified www.biotechlab.nwu.edu/ pe/Sld022.htm

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  www.biotechlab.nwu.edu/ pe/Sld022.htm

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  www.biotechlab.nwu.edu/ pe/Sld022.htm

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  Plateau effect in PCR reaction

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  www.biotechlab.nwu.edu/ pe/Sld022.htm Plateau effect in PCR reaction

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  Non-specific PCR and how to improve it

  Just PCR 5% D M S O D M S O + G L Y M A R K E R Decrease in Mg concentraton

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  PCR enzymes

  Taq DNA polymerase, the first enzyme used for PCR, is still the most popular. -- high processivity -- is the least expensive choice -- generates PCR products with single A overhangs on the 3´-ends (Suitable for TOPO-cloning) “Topo” cloning system (Invitrogen) Half-life at 95C is 1.6 hours

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  Tth polymerase

  Thermus thermophilus strain HB8. RNA-dependent DNA-polymerase activity in the presence of Mn2+ ions. DNA-dependent DNA-polymerase activity in the presence of Mg2+ ions. The fragment should be ideally smaller 1 kb. Mn 2+ Mg 2+

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  Pfu polimerase

  Proofreading or high fidelity DNA polymerases (from Pyrococcus furiosus). approx.1 / 2, 000,000 nucleotides before making an error. In comparison Taq DNA polymerase makes an error in approx. every 1/ 10,000 nucleotides. can tolerate temperatures exceeding 95°C, enabling it to PCR amplify GC-rich targets. more expensive

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  Pol Vent (From Thermococcus litoralis)

  also known as Tli polymerase Very termostable: Half-life at 95 C is approximately 7 hours Vent error rate is intermediate between Taq and Pfu. 2-5 x 10-5 errors/bp 3'->5' exonuclease activity presents Other polymerases: Deep Vent (Pyrococcus species GB-D) (New England Biolabs) New England Biolabs claims fidelity is equal to or greater than that of Vent. Replinase(Thermus flavis) 1.03 x 10-4 errors/base

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  Long-Range PCR

  Use of two polymerases: a non-proofreading polymerase Taq is the main polymerase in the reaction, a proofreading polymerase (3' to 5' exo) Pwo is present at a lower concentration. 22-24 kb PCR products are achieved on Qiagen and Eppendorf PCR mixes Taq+ Pwo (Pyrococcus woesei) ; Pwo is very stable, 2 hrs at 100 C

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  3. SEQUENCING: (Sanger method)

  Sanger method: http://www.kids-dna.com/dnatube.gif Frederick Sanger (Nobel prize 1980 with Paul Berg and Walter Gilbert)

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  Dideoxynucleotide blocks chain elongation

  http://www.cbs.dtu.dk/staff/dave/roanoke/fig5_38.jpg

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  DNA sequencing: chemistry, with terminator dyes

  * * * * * * * * * * * * * *

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  Semi-automated fluorescent DNA sequencing:

  template + polymerase + dCTP dTTP dGTP dATP ddATP ddGTP ddTTP ddCTP extension electrophoresis A•T G•C A•T T•A C•G T•A G•C G•C A•T G•C T•A T•A C•G T•A G•C A•T

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  Cycle Sequencing - PCR

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  Applied Biosystems Inc., have designed an automated method that combines the PCR and actual sequencing

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  DNA sequencing by primer walking

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  Chemical synthesis of DNA

  Chain grows: 3’-> 5’

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  General consideration about Gene Expression

  Expression Host -> Expression System Promoter system -> expression vector Properties of product -> stability Production level

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  Comparison of expression systems

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  Use of Lac promoter (pLac) for expression of foreign protein

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  Prokaryotic Expression vector

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  PstI T7/lac phoA cutinase NarI SalI HindIII BamHI phoA-cut NdeI S/D Term pFCEX1

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  Eukaryotic Expression vector

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  Promoters

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  Control of transcription of the lac operon.

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  Terminators

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  Northern Blot -> to study transcription level

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  Expression studies by microarray technique