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Biochemical Tool

Biochemical Tool. Electrophoresis Hybridization. E. v. F. + - + - - -. -. +. q. f. Electrophoresis. Electro = flow of electricity, Phoresis= to carry across (from the Greek). Molecules are separated by electric force F = qE : where q is net charge, E is electric field strength

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Biochemical Tool

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  1. Biochemical Tool Electrophoresis Hybridization

  2. E v F + - + - - - - + q f Electrophoresis • Electro = flow of electricity, • Phoresis= to carry across (from the Greek) • Molecules are separated by electric force • F = qE : where q is net charge, E is electric field strength • The velocity is encountered by friction • qE = fv : where f is frictional force, v is velocity • Therefore, mobility per unit field (U) = v/q = q/f = q/6pr : where  is viscosity of supporting medium, r is radius of sphere molecule

  3. + Definition The separation of charged molecules using their different rates of migration in an electrical field - FACTORS INFLUENCING SEPARATION Samples • Charge Density on Molecules - Difference between pH Separating Gel • Molecular Size and Shape

  4. Electrophoresis - • Factors affected the mobility of molecules • 1. Molecular factors • Charge • Size • Shape • 2. Environment factors • Electric field strength • Supporting media (pore: sieving effect) • Running buffer +

  5. Types of supporting media • Paper • Agarose gel (Agarose gel electrophoresis) • Polyacrylamide gel (PAGE) • pH gradient (Isoelectric focusing electrophoresis) • Cellulose acetate

  6. Gel electrophoresis • A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatin • Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied • Charged particles can include DNA, amino acids, peptides

  7. Poliakrialimida • Polimer dari akrilamid • Pori-porinya lebih kecil dari polimer agarosa • Menghasilkan tingkat resolusi yang lebih tinggi • Gel dibuat dengan menggunakan 2 lembaran kaca atau plastik mika

  8. Poliakrialimida • Penyangga: TBE • Kegunaan: • Memisahkan DNA berukuran kecil (AFLP, SNP) • mengurutkan DNA • Memisahkan protein (perlu ditambah SDS, sehingga disebut SDS-PAGE: SDS poly acrylamide Gel Electrophoresis)

  9. Pembuatan gel poliakrialimida • Akrilamida + metilen bis akrilamida • Ukuran pori ditentukan dengan menentukan konsentrasi akrilamida dan metilen bis akrilamidanya

  10. Electrophoresis • Polyacrylamide Gels • Acrylamide polymer; very stable gel • can be made at a wide variety of concentrations • gradient of concentrations: large variety of pore sizes (powerful sieving effect)

  11. Electrophoresis SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) • Sodium Dodecyl Sulfate = Sodium Lauryl Sulfate: CH3(CH2)11SO3- Na+ • Amphipathic molecule • Strong detergent to denature proteins • Binding ratio: 1.4 gm SDS/gm protein • Charge and shape normalization

  12. Electrophoresis • Isoelectric Focusing Electrophoresis (IFE) • Separate molecules according to their isoelectric point (pI) • At isoelectric point (pI) molecule has no charge (q=0), hence molecule ceases • pH gradient medium

  13. Electrophoresis • 2-dimensional Gel Electrophoresis • First dimension is IFE (separated by pI) • Second dimension is SDS-PAGE (separated by size) • So called 2D-PAGE • High throughput electrophoresis, high resolution • Core methods for “Proteomics”

  14. 2-dimensional Gel Electrophoresis • Spot coordination • pI • MW

  15. 2-dimensional Gel Electrophoresis Application

  16. Hybridization and Blotting

  17. Hybridization

  18. Hybridization • Can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily degraded) • Dependent on the extent of complementation • Dependent on temperature, salt concentration, and solvents • Small changes in the above factors can be used to discriminate between different sequences (e.g., small mutations can be detected) • Probes can be labeled with radioactivity, fluorescent dyes, enzymes, etc. • Probes can be isolated or synthesized sequences

  19. Oligonucleotide probes • Single stranded DNA (usually 15-40 bp) • Degenerate oligonucleotide probes can be used to identify genes encoding characterized proteins • Use amino acid sequence to predict possible DNA sequences • Hybridize with a combination of probes • TT(T/C) - TGG - ATG - GA(T/C) - TG(T/C) - could be used for FWMDC amino acid sequence • Can specifically detect single nucleotide changes

  20. Detection of Probes • Probes can be labeled with radioactivity, fluorescent dyes, enzymes. • Radioactivity is often detected by X-ray film (autoradiography) • Fluorescent dyes can be detected by fluorometers, scanners • Enzymatic activities are often detected by the production of dyes or light (x-ray film)

  21. RNA Blotting (Northerns) • RNA is separated by size on a denaturing agarose gel and then transferred onto a membrane (blot) • Probe is hybridized to complementary sequences on the blot and excess probe is washed away • Location of probe is determined by detection method (e.g., film, fluorometer)

  22. Applications of RNA Blots • Detect the expression level and transcript size of a specific gene in a specific tissue or at a specific time. Sometimes mutations do not affect coding regions but transcriptional regulatory sequences (e.g., UAS/URS, promoter, splice sites, copy number, transcript stability, etc.)

  23. Western Blot • Highly specific qualitative test • Can determine if above or below threshold • Typically used for research • Use denaturing SDS-PAGE • Solubilizes, removes aggregates & adventitious proteins are eliminated Components of the gel are then transferred to a solid support or transfer membrane weight Paper towel Wet filter paper Transfer membrane Paper towel

  24. Add antibody against yours with a marker (becomes the antigen) Stain the bound antibody for colour development It should look like the gel you started with if a positive reaction occurred Western Blot Rinse with ddH2O • Block membrane e.g. dried nonfat milk Add monoclonal antibodies Rinse again Antibodies will bind to specified protein

  25. Polymerase Chain Reaction(PCR)

  26. PCR • A simple rapid, sensitive and versatile in vitro method for selectively amplifying defined sequences/regions of DNA/RNA from an initial complex source of nucleic acid - generates sufficient for subsequent analysis and/or manipulation • Amplification of a small amount of DNA using specific DNA primers (a common method of creating copies of specific fragments of DNA) • DNA fragments are synthesized in vitro by repeated reactions of DNA synthesis (It rapidly amplifies a single DNA molecule into many billions of molecules) • In one application of the technology, small samples of DNA, such as those found in a strand of hair at a crime scene, can produce sufficient copies to carry out forensic tests. • Each cycle the amount of DNA doubles

  27. Background on PCR • Ability to generate identical high copy number DNAs made possible in the 1970s by recombinant DNA technology (i.e., cloning). • Cloning DNA is time consuming and expensive • Probing libraries can be like hunting for a needle in a haystack. • Requires only simple, inexpensive ingredients and a couple hours.

  28. Background on PCR • PCR, “discovered” in 1983 by Kary Mullis DNA template Primers (anneal to flanking sequences) DNA polymerase dNTPs Mg2+ Buffer • Can be performed by hand or in a machine called a thermal cycler. • 1993: Nobel Prize for Chemistry

  29. Three Steps • Separation: Double Stranded DNA is denatured by heat into single strands. • Short Primers for DNA replication are added to the mixture. • DNA polymerase catalyzes the production of complementary new strands. • Copying: the process is repeated for each new strand created • All three steps are carried out in the same vial but at different temperatures

  30. Step 1: Separation • Combine Target Sequence, DNA primers template, dNTPs, Taq Polymerase • Target Sequence: Usually fewer than 3000 bp • Identified by a specific pair of DNA primers- usually oligonucleotides that are about 20 nucleotides • Heat to 95°C to separate strands (for 0.5-2 minutes) • Longer times increase denaturation but decrease enzyme and template

  31. Magnesium as a Cofactor • Stabilizes the reaction between: • oligonucleotides and template DNA • DNA Polymerase and template DNA

  32. Heat: Denatures DNA by uncoiling the Double Helix strands.

  33. Step 2: Priming • Decrease temperature by 15-25 ° • Primers anneal to the end of the strand • 0.5-2 minutes • Shorter time increases specificity but decreases yield • Requires knowledge of the base sequences of the 3’ - end

  34. Selecting a Primer • Primer length • Melting Temperature (Tm) • Specificity • Complementary Primer Sequences • G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches • 3’-end Sequence • Single-stranded DNA

  35. Step 3: Polymerization • Since the Taq polymerase works best at around 75 ° C (the temperature of the hot springs where the bacterium was discovered), the temperature of the vial is raised to 72-75 °C • The DNA polymerase recognizes the primer and makes a complementary copy of the template which is now single stranded. • Approximately 150 nucleotides/sec

  36. Potential Problems with Taq • Lack of proof-reading of newly synthesized DNA. • Potentially can include di-Nucleotriphosphates (dNTPs) that are not complementary to the original strand. • Errors in coding result • Recently discovered thermostable DNA polymerases, Tth and Pfu, are less efficient, yet highly accurate.

  37. How PCR works: Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence. Next, denature the DNA at 94˚C. Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA. Extend primers at 70-75˚C using a heat-resistant DNA polymerase (e.g., Taq polymerase derived from Thermus aquaticus). Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (220) to 35 trillion copies (245) of the target DNA. Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely. Cool to 4˚C and store or use amplified PCR product for analysis.

  38. Example thermal cycler protocol used in lab: Step 1 7 min at 94˚C Initial Denature Step 2 45 cycles of: 20 sec at 94˚C Denature 20 sec at 64˚C Anneal 1 min at 72˚C Extension Step 3 7 min at 72˚C Final Extension Step 4 Infinite hold at 4˚C Storage

  39. The Polymerase Chain Reaction

  40. PCR amplification • Each cycle the oligo-nucleotide primers bind most all templates due to the high primer concentration • The generation of mg quantities of DNA can be achieved in ~30 cycles (~ 4 hrs)

  41. OPTIMISING PCR – THE REACTION COMPONENTS • Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen • Thermo-stable DNA polymerase e.g. Taq polymerase • Oligonucleotides Design them well! • Buffer Tris-HCl (pH 7.6-8.0) Mg2+dNTPs (dATP, dCTP, dGTP, dTTP)

  42. RAW MATERIAL • Tissue, cells, blood, hair root, saliva, semen • Obtain the best starting material you can. • Some can contain inhibitors of PCR, so they must be removed e.g. Haem in blood • Good quality genomic DNA if possible • Blood – consider commercially available reagents Qiagen– expense? • Empirically determine the amount to add

  43. POLYMERASE • Number of options available • Taq polymerase Pfu polymerase Tth polymerase • How big is the product? • 100bp 40-50kb • What is end purpose of PCR?1. Sequencing - mutation detection-. Need high fidelity polymerase • -. integral 3’ 5' proofreading exonuclease activity • 2. Cloning

  44. PRIMER DESIGN • Length ~ 18-30 nucleotides (21 nucleotides) • Base composition; 50 - 60% GC richpairs should have equivalent Tms • Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C] • Initial use Tm–5°C • Avoid internal hairpin structuresno secondary structure • Avoid a T at the 3’ end • Avoid overlapping 3’ ends – will form primer dimers • Can modify 5’ ends to add restriction sites

  45. PRIMER DESIGN Use specific programs OLIGOMedprobe PRIMERDESIGNERSci. Ed software Also available on the internet http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html

  46. 1 1.5 2 2.5 3 3.5 4 mM Mg2+ CONCENTRATION Normally, 1.5mM MgCl2 is optimal Best supplied as separate tube Always vortex thawed MgCl2 Mg2+ concentration will be affected by the amount of DNA, primers and nucleotides

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