1. Reminder: All molecular techniques are based on the chemical “personality” (or chemical properties) of the DNA molecule (or nucleic acids)

2. Cellular level Organelle level Molecular level: Macromolecules Atomic level C, H, O, N, S, P Microscope Cell fractionation -Nucleus -Mitochondria -RER, cell membrane -SER -Cytosol ProteinsCarbohydratesLipidsNucleic acids Studies of cell -Fractionation -Purification/ Identification -Structure/ Function

3. CONTENTS Electrophoresis Blotting and Hybridization Polymerase Chain Reaction DNA Sequences

4. Negatively-charged phosphate-sugar backbone - - - - Hydrogen bonds Specificity of nucleotides Various lengths

5. DNA GEL ELECTROPHORESIS 1.For separating DNA strands of any size/length 2.Uses a gel to separate DNA strands 3.Uses electricity

6. 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 + - + - - - -+ E F f v q Electrophoresis

7. 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 - + Electrophoresis

9. Types of supporting media  Paper  Agarose gel (Agarose gel electrophoresis)  Polyacrylamide gel (PAGE)  pH gradient (Isoelectric focusing electrophoresis)  Cellulose acetate Electrophoresis

10. Agarose:  purified large MW polysaccharide (from agar)  very open (large pore) gel  used frequently for large DNA molecules Agarose Gel

11. Agarose gel staining Ethidium bromide

12. 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) Electrophoresis

13.  Sodium Dodecyl Sulfate = Sodium Lauryl Sulfate: CH 3 (CH 2 ) 11 SO 3 - Na +  Amphipathic molecule  Strong detergent to denature proteins  Binding ratio: 1.4 gm SDS/gm protein  Charge and shape normalization SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

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

15. -First dimension is IFE (separated by charge) -Second dimension is SDS- PAGE (separated by size) -So called 2D-PAGE -High throughput electrophoresis, high resolution 2-dimensional Gel Electrophoresis

16. Spot coordination pH MW

17. Hybridization and Blotting

18. Hybridization Pairing of complementary DNA and/or RNA

19. Hybridization It can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily degraded) It depended on the extent of complementation It depended 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. Probes can be isolated or synthesized sequences

20. 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

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

22. RNA Blotting (Northerns) RNA is separated by size on a denaturing agarose gel and then transferred onto a membrane (blot) 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 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 ) Location of probe is determined by detection method (e.g., film, fluorometer )

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

24. Western Blot Add monoclonal antibodies Rinse again Antibodies will bind to specified protein Stain the bound antibody for colour development It should look like the gel you started with if a positive reaction occurred Block membrane e.g. dried nonfat milk Block membrane e.g. dried nonfat milk Rinse with ddH 2 O Add antibody against yours with a marker (becomes the antigen)

25. Polymerase Chain Reaction (PCR)

26. 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 PCR

27. The 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 PCR, “discovered” in 1983 by Kary Mullis, Nobel Prize for Chemistry (1993). It can be performed by hand or in a machine called a thermal cycler. Background on PCR

28. Three Steps Separation Double Stranded DNA is denatured by heat into single strands. Short Primers for DNA replication are added to the mixture. Priming 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

29. Step 1: Separation Combine Target Sequence, DNA primers template, dNTPs, Taq Polymerase Target Sequence 1. Usually fewer than 3000 bp 2. 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 Magnesium as a Cofactor Mg stabilizes the reaction between: oligonucleotides and template DNA DNA Polymerase and template DNA

30. Heat Denatures DNA by uncoiling the Double Helix strands.

31. Step 2: Priming Decrease temperature by 15-25 °C 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

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

33. 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 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. The DNA polymerase recognizes the primer and makes a complementary copy of the template which is now single stranded. Approximately 150 nucleotides/sec Approximately 150 nucleotides/sec

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

35. 1.Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence. 2.Next, denature the DNA at 94˚C. 3.Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA. 4.Extend primers at 70-75˚C using a heat-resistant DNA polymerase (e.g., Taq polymerase derived from Thermus aquaticus). 5.Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (2 20 ) to 35 trillion copies (2 45 ) of the target DNA. 6.Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely. 7. Cool to 4˚C and store or use amplified PCR product for analysis. How PCR works

36. Step 1 7 min at 94˚CInitial Denature Step 2 45 cycles of: 20 sec at 94˚CDenature 20 sec at 64˚CAnneal 1 min at 72˚CExtension Step 3 7 min at 72˚CFinal Extension Step 4 Infinite hold at 4˚CStorage Thermal cycler protocol Example

37. The Polymerase Chain Reaction

41. 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)

42. Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, semen Thermo-stable DNA polymerase e.g. Taq polymerase Oligonucleotides Design them well! Buffer Tris-HCl (pH 7.6-8.0) Mg 2+ dNTPs (dATP, dCTP, dGTP, dTTP) OPTIMISING PCR THE REACTION COMPONENTS

43. Organims, Organ, Tissue, cells ( blood, hair root, semen, callus, leaves, root, seed) Obtain the best starting material. Some can contain inhibitors of PCR, so they must be removed e.g. Haem in blood Good quality genomic DNA if possible Empirically determine the amount to add RAW MATERIAL

44. 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 3. Marker development POLYMERASE

45. Length ~ 10-30 nucleotides (21 nucleotides for gene isolation) Base composition: 50 - 60% GC rich, pairs 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 structures No 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 PRIMER DESIGN

46. Use specific programs OLIGO Medprobe PRIMER DESIGNER Sci. Ed software Also available on the internet http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html

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

48. USE MASTERMIXES WHERE POSSIBLE

49. How Powerful is PCR? PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. The template DNA need not be highly purified — a boiled bacterial colony. The template DNA need not be highly purified — a boiled bacterial colony. The PCR product can be digested with restriction enzymes, sequenced or cloned. The PCR product can be digested with restriction enzymes, sequenced or cloned. PCR can amplify a single DNA molecule, e.g. from a single sperm. PCR can amplify a single DNA molecule, e.g. from a single sperm.

50. Applications of PCR Amplify specific DNA sequences (genomic DNA, cDNA, recombinant DNA, etc.) for analysis Amplify specific DNA sequences (genomic DNA, cDNA, recombinant DNA, etc.) for analysis 1. Gene isolation 2. Fingerprint development Introduce sequence changes at the ends of fragments Introduce sequence changes at the ends of fragments Rapidly detect differences in DNA sequences (e.g., length) for identifying diseases or individuals Rapidly detect differences in DNA sequences (e.g., length) for identifying diseases or individuals Identify and isolate genes using degenerate oligonucleotide primers Identify and isolate genes using degenerate oligonucleotide primers Design mixture of primers to bind DNA encoding conserved protein motifsDesign mixture of primers to bind DNA encoding conserved protein motifs Genetic diagnosis - Mutation detection The basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10 -9 bp Genetic diagnosis - Mutation detection The basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10 -9 bp

51. Paternity testing Mutagenesis to investigate protein function Quantify differences in gene expression → Reverse transcription (RT)-PCR Identify changes in expression of unknown genes → Differential display (DD)-PCR Forensic analysis at scene of crime Industrial quality control DNA sequencing Applications of PCR

53. DNA Sequencing

54. DNA sequencing Determination of nucleotide sequence Determination of nucleotide sequence  the determination of the precise sequence of nucleotides in a sample of DNA Two similar methods: Two similar methods: 1. Maxam and Gilbert method 2. Sanger method They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in length by a single nucleotide at the other end They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in length by a single nucleotide at the other end This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands determined This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands determined

55. The Maxam-Gilbert Technique Principle: Principle: Chemical Degradation of Purines Purines (A, G) damaged by dimethylsulfatePurines (A, G) damaged by dimethylsulfate Methylation of baseMethylation of base Heat releases baseHeat releases base Alkali cleaves GAlkali cleaves G Dilute acid cleave A>GDilute acid cleave A>G

56. Maxam-Gilbert Technique Pyrimidines (C, T) are damaged by hydrazinePyrimidines (C, T) are damaged by hydrazine Piperidine cleaves the backbonePiperidine cleaves the backbone 2 M NaCl inhibits the reaction with T2 M NaCl inhibits the reaction with T

57. Maxam and Gilbert Method Chemical degradation of purified fragments (chemical degradation) Chemical degradation of purified fragments (chemical degradation) The single stranded DNA fragment to be sequenced is end-labeled by treatment with alkaline phosphatase to remove the 5’phosphate The single stranded DNA fragment to be sequenced is end-labeled by treatment with alkaline phosphatase to remove the 5’phosphate It is then followed by reaction with P-labeled ATP in the presence of polynucleotide kinase, which attaches P labeled to the 5’terminal It is then followed by reaction with P-labeled ATP in the presence of polynucleotide kinase, which attaches P labeled to the 5’terminal The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base 1. Aliquot A + dimethyl sulphate, which methylates guanine residue 2. Aliquot B + formic acid, which modifies adenine and guanine residues 3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified

58. Maxam-Gilbert sequencing - modifications

59. Maxam-Gilbert sequencing: Summary

60. Advantages/disadvantages Maxam-Gilbert sequencing Requires lots of purified DNA, and many intermediate purification steps Relatively short readings Automation not available (sequencers) Remaining use for ‘footprinting’ (partial protection against DNA modification when proteins bind to specific regions, and that produce ‘holes’ in the sequence ladder) In contrast, the Sanger sequencing methodology requires little if any DNA purification, no restriction digests, and no labeling of the DNA sequencing template

61. Sanger Fred Sanger, 1958 Fred Sanger, 1958 Was originally a protein chemistWas originally a protein chemist Made his first mark in sequencing proteinsMade his first mark in sequencing proteins Made his second mark in sequencing RNAMade his second mark in sequencing RNA 1980 dideoxy sequencing 1980 dideoxy sequencing

62. Original Sanger Method Random incorporation of a dideoxynucleoside triphosphate into a growing strand of DNA Random incorporation of a dideoxynucleoside triphosphate into a growing strand of DNA Requires DNA polymerase I Requires DNA polymerase I Requires a cloning vector with initial primer (M13, high yield bacteriophage, modified by adding: beta- galactosidase screening, polylinker) Requires a cloning vector with initial primer (M13, high yield bacteriophage, modified by adding: beta- galactosidase screening, polylinker) Uses 32 P-deoxynucleoside triphosphates Uses 32 P-deoxynucleoside triphosphates

63. Sanger Method in-vitro DNA synthesis using ‘terminators’, use of dideoxi- nucleotides that do not permit chain elongation after their integration in-vitro DNA synthesis using ‘terminators’, use of dideoxi- nucleotides that do not permit chain elongation after their integration DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific nucleotides DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific nucleotides Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system Incorporation of di-deoxynucleotides into growing strand terminates synthesis Incorporation of di-deoxynucleotides into growing strand terminates synthesis Synthesized strand sizes are determined for each di- deoxynucleotide by using gel or capillary electrophoresis Synthesized strand sizes are determined for each di- deoxynucleotide by using gel or capillary electrophoresis Enzymatic methods Enzymatic methods

64. Dideoxynucleotide no hydroxyl group at 3’ end prevents strand extension CH2 O OPPP 5’ 3’ BASE

65. The principles Partial copies of DNA fragments made with DNA polymerase Collection of DNA fragments that terminate with A,C,G or T using ddNTP Separate by gel electrophoresis Read DNA sequence

66. CCGTAC 3’ 5’ 3’ primer dNTP ddATP GGCA ddTTP GGCAT ddCTP GGCG ddGTP G GGCATG A T C G

67. Chain Terminator Basics Target Template-Primer Extend ddA ddG ddC ddT Labeled Terminators ddA AddC ACddG ACGddT TGCA dN : ddN 100 : 1

69. Electrophoresis

70. Sanger Method Sequencing Gel

71. Sequencing of DNA by the Sanger method

72. Comparison Sanger Method Sanger Method EnzymaticEnzymatic Requires DNA synthesisRequires DNA synthesis Termination of chain elongationTermination of chain elongation Maxam Gilbert Method Maxam Gilbert Method Chemical Requires DNA Requires long stretches of DNA Breaks DNA at different nucleotides