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  4. 4.  DNA sequencing is the determination of the order of bases insample of DNA. It is the reading of the genetic code. However, not all DNA sequences are genes (i.e. coding regions)as there may, depending on the organism and the source of theDNA sample, also be promoters, tandem repeats, introns, etc. 4
  5. 5. Two methods for the large-scale sequencing of DNA becameavailable in the late 1970s.Both based on generation of DNA fragments of differentlengths which start at a fixed point and terminate at specificnucleotides.DNA fragments are separated by size on polyacrylamidegels and the nucleotide sequences are directly read from thegel. 5
  6. 6. 1. Maxam-Gilbert sequencing (chemical cleavage methodusing double-stranded (ds) DNA) in which the sequence of adouble-stranded DNA molecule is determined by treatmentwith chemicals that cut the molecule at specific nucleotidepositions. 6
  7. 7. 2. Sanger-Coulson sequencing (chain termination methodusing single-stranded (ss) DNA) in which the sequence of asingle-stranded DNA molecule is determined by enzymaticsynthesis of complementary polynucleotide chains, these chainsterminating at specific nucleotide positions. 7
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  9. 9. Sanger Method of DNA SequencingMajor steps1. Template DNA (ssDNA)2. Primer annealing3. Complementary strand synthesis4. Labeling for the detection of fragments5. Chain termination using ddNTPs6. Resolution on denaturing PAGE7. Visualization of bands by autoradiography 9
  10. 10.  preparation of identical single-stranded DNA molecules.The first step is to anneal a short oligonucleotide to the sameposition on each molecule, this oligonucleotide subsequently actingas the primer for synthesis of a new DNA strand that iscomplementary to the template which is to be sequenced .The strand synthesis reaction catalyzed by a DNA polymeraseenzyme and requires the four deoxyribonucleotide triphosphates(dNTPs - dATP, dCTP, dGTP and dTTP) as substrates, wouldnormally continue until several thousand nucleotides had beenpolymerized. 10
  11. 11. This does not occur in a chain termination sequencingexperiment because, as well as the four dNTPs, a small amountof a dideoxynucleotide (e.g. ddATP) is added to the reaction. The polymerase enzyme does not discriminate betweendNTPs and ddNTPs, so the dideoxynucleotide can beincorporated into the growing chain, but it then blocks furtherelongation because it lacks the 3′-hydroxyl group needed toform a connection with the next nucleotide 11
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  15. 15. Reading a DNA Sequencing Gel Sequence 5’ to 3’ C G G G C G T 15
  16. 16. The smallest fragments will be at the bottom of the gel, thelargest fragments at the top.The DNA sequence can be determined by determining theterminating base for the shortest fragment, then for the nextshortest fragment for all of the DNA fragments 16
  17. 17. MAXAM-GILBERT SEQUENCINGThis chemical cleavage method uses double-stranded DNAsamples and so does not require cloning of DNA into an M13phage vector to produce single-stranded DNA. It involves modification of the bases in DNA followed bychemical base-specific cleavage.Stages:1. Double-stranded DNA to be sequenced is labeled byattaching a radioactive phosphorus (32P) group to the 5 end.Polynucleotide kinase enzyme and 32P-dATP is used here. 17
  18. 18. 2. Using dimethyl sulphoxide (DMSO) and heating to90oC, the two strands of the DNA are separated and purified(e.g. using gel electrophoresis and the principle that one ofthe strands is likely to be heavier than the other due to thefact that it contains more purine nucleotides (A and G) thanpyrimidines (C and T) which are lighter.3. Single-stranded sample is split into separate samplesand each is treated with one of the cleavage reagents. Thispart of the process involves alteration of bases (e.g.dimethylsulphate methylates guanine) followed by removalof altered bases. Lastly, piperidine is used for cleavage ofthe strand at the points where bases are missing 18
  19. 19. Chemical Chemical Base Chemical used for used for used forspecificity base alteration altered base strand removal cleavage G Dimethylsulphate Piperidine Piperidine A+G Acid Acid Piperidine C+T Hydrazine Piperidine Piperidine Hydrazine + High C Piperidine Piperidine salt A>C Alkali Piperidine Piperidine 19
  20. 20. G A+G C +T C Sequence C G T T C C G G A C T A A 20
  21. 21. Automated DNA Sequencing with Fluorescent DyesEach different ddNTP is coupled to a different coloredfluorescent dye ddTTP is red; ddGTP is black etc. 21
  22. 22. Alternative Sequencing Methods: PyrosequencingPyrosequencing is based on the generation of light signalthrough release of pyrophosphate (PPi) on nucleotideaddition. DNAn + dNTP  DNAn+1 + PPIPPi is used to generate ATP from adenosine phosphosulfate(APS). APS + PPI  ATPATP and luciferase generate light by conversion of luciferin tooxyluciferin. 22
  23. 23.  Each nucleotide is added in turn. Only one of four will generate a light signal. The remaining nucleotides are removed enzymatically. The light signal is recorded on a pyrogram. DNA sequence: A T C A GG CC T Nucleotide added : A T C A G C T 23
  24. 24. Bisulfite Sequencing Bisulfite sequencing is used to detect methylation in DNA. Bisulfite deaminates cytosine, making uracil. Methylated cytosine is not changed by bisulfite treatment. The bisulfite-treated template is then sequenced. 24
  25. 25. Bisulfite Sequencing The sequence of treated and untreated templates is compared. Me Me Me Methylated sequence: GTC GGC GATCTATC GTGCA … Me Me Me Treated sequence: GTC GGC GATUTATC GTGUA … DNA Sequence: (Untreated) reference: ...GTCGGCGATCTATCGTGCA… Treated sequence: ...GTCGGCGATUTATCGTGUA… This sequence indicates that these Cs are methylated. 25
  26. 26. Genome sequencing strategies Only short DNA molecules (~800 bp) can be sequenced in one read, so large DNA molecules, such as genomes, longer sequences must be subdivided into smaller fragments and subsequently reassembled to give the overall sequence. Genome sequencing can be approached in two ways 26
  27. 27. Whole-genome shotgun sequencingThe whole-genome shotgun approach was firstproposed by Craig Venter and colleagues as ameans of speeding up the acquisition ofcontiguous sequence data for large genomessuch as the human genome and those of othereukaryotes (Venter et al., 1998; Marshall 27
  28. 28. Clone contig sequencing: Involves the systematic production and sequencing of sub clones arrange overlapping clones before sequencing. 28
  29. 29. DNA MODIFICATION & RESTRICTION Bacteria can destroy an invading or foreign DNA from an otherspecies, thus preventing its replication, transcription, orincorporation in to the host cell genome.This is made possible by an ingenious combination of twoenzymatic processes called modification & restriction. 29
  30. 30. MODIFICATION 30
  31. 31. MODIFICATION It is the enzymatic alteration of its own DNA by the cell, in a species distinctive way , thus differentiating it from that of other species. The protective modification of the host cell DNA is brought about by modification methylases,which methylate certain adenine residues. Once the host cell DNA is modified in this manner,it cannot be degraded by that cells restriction enzymes. 31
  32. 32.  The restriction methylases transfer methyl groups from s-adenosylmethionine to pairs of adenine residues in duplex DNA , one in each strand; the two adenine are on adjacent or near by base pairs. The sequence of bases on the two stands between and near the methylated adenines is symmetrical on either side of a mid point. 32
  33. 33. DNA RESTRICTION 33
  34. 34. A Restriction Enzyme (or restriction endonuclease) is anenzyme that cuts double-stranded DNA at specificrecognition nucleotide sequences known asrestrictionsites. Inside a bacterial host, the restriction enzymes selectivelycut up foreign DNA in a process called restrication. To cut the DNA, a restriction enzyme makes twoincisions, once through each sugar-phosphate backbone(i.e. each strand) of the DNA double helix. 34
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  36. 36. Restriction Enzymes scan theDNA sequence. 36
  37. 37.  recognition site 5-GTATAC-3 :::::: 3-CATATG-5 A palindromic recognition site reads the same on the reverse strand as it does on the forward strand when both are read in the same orientation. Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in theDNA. There are two types of palindromic sequences that can be possible in DNA. 37
  38. 38. The Mirror like palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backwards on a single strand of DNA strand, as in GTAATG. The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (i.e., of double-stranded DNA), as in GTATAC (GTATAC being complementary to CATATG). Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes. 38
  39. 39.  Different restriction enzymes that recognize the same sequence are known as neoschizomers. These often cleave in different locales of the sequence. Different enzymes that recognize and cleave in the same location are known as isoschizomers. Types Restriction endonucleases are categorized into three or four general groups (Types I, II and III) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence. 39
  40. 40.  There are four classes of restriction endonucleases: types I, II,III and IV. All types of enzymes recognise specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5- phosphates They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements 40
  41. 41.  Type I restriction enzymes were the first to be identified and were first identified in two different strains (K-12 and B) of E. coli. These enzymes cut at a site that differs, and is a random distance (at least 1000 bp) away, from their recognition site. Cleavage at these random sites follows a process of DNA translocation, which shows that these enzymes are also molecular motors. The recognition site is asymmetrical and is composed of two specific portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides— separated by a non-specific spacer of about 6–8 nucleotides. 41
  42. 42.  These enzymes are multifunctional and are capable of both restriction and modification activities, depending upon the methylation status of the target DNA. The cofactors S-Adenosyl methionine (AdoMet), hydrolyzed adenosine triphosphate (ATP), and magnesium (Mg2+) ions, are required for their full activity. Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; 42
  43. 43.  HsdR is required for restriction; HsdM is necessary for adding methyl groups to host DNA (methyltransferase activity) and HsdS is important for specificity of the recognition (DNA-binding) site in addition to both restriction (DNA cleavage) and modification (DNA methyltransferase) activity.[ 43
  44. 44. Type II: 44
  45. 45.  They are a dimer of only one type of subunit; their recognition sites are usually undivided and palindromic and 4–8 nucleotides in length, they recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity—they usually require only Mg2+ as a cofactor.[ These are the most commonly available and used restriction enzymes. 45
  46. 46.  In the 1990s and early 2000s, new enzymes from this family were discovered that did not follow all the classical criteria of this enzyme class, and new subfamily nomenclature was developed to divide this large family into subcategories based on deviations from typical characteristics of type II enzymes. 46
  47. 47.  Type IIB restriction enzymes (e.g. BcgI and BplI) are multimers, containing more than one subunit They cleave DNA on both sides of their recognition to cut out the recognition site. They require both AdoMet and Mg2+ cofactors. Type IIE restriction endonucleases (e.g. NaeI) cleave DNA following interaction with two copies of their recognition sequence.[ One recognition site acts as the target for cleavage, while the other acts as an allosteric effector that speeds up or improves the efficiency of enzyme cleavage. 47
  48. 48.  Type IIG restriction endonucleases (Eco57I) do have a single subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active. Type IIM restriction endonucleases, such as DpnI, are able to recognize and cut methylated DNA Type IIS restriction endonucleases (e.g. FokI) cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites These enzymes may function as dimers. Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits 48
  49. 49.  Type III restriction enzymes (e.g. EcoP15) recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site. These enzymes contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA methylation and restriction, respectively. They are components of prokaryotic DNA restriction- modification mechanisms that protect the organism against invading foreign DNA. 49
  50. 50.  Type III enzymes are hetero- oligomeric, multifunctional proteins composed of two subunits, Res and Mod. The Mod subunit recognises the DNA sequence specific for the system and is a modification methyltransferase; as such it is functionally equivalent to the M and S subunits of type I restriction endonuclease. Res is required for restriction, although it has no enzymatic activity on its own. 50
  51. 51.  Type III enzymes recognise short 5-6 bp long asymmetric DNA sequences and cleave 25-27 bp downstream to leave short, single-stranded 5 protrusions They require the presence of two inversely oriented unmethylated recognition sites for restriction to occur. These enzymes methylate only one strand of the DNA, at the N-6 position of adenosyl residues, so newly replicated DNA will have only one strand methylated, which is sufficient to protect against restriction. 51
  52. 52.  Type III enzymes belong to the beta-subfamily of N6 adenine methyltransferases, containing the nine motifs that characterize this family, including motif I, the AdoMet binding pocket (FXGXG), and motif IV, the catalytic region (S/D/N (PP) Y/F).[ 52
  53. 53. 5GGTACC 5---GGTAC C---3 Klebsiella pneumoniae 3CCATGG 3---C CATGG---5 5CTGCAG 5---CTGCA G---3PstI[48] Providencia stuartii 3GACGTC 3---G ACGTC---5 Streptomyces 5GAGCTC 5---GAGCT C---3SacI[48] achromogenes 3CTCGAG 3---C TCGAG---5 5GTCGAC 5---G TCGAC---3SalI[48] Streptomyces albus 3CAGCTG 3---CAGCT G---5 Streptomyces 5AGTACT 5---AGT ACT---3ScaI[48] caespitosus 3TCATGA 3---TCA TGA---5 5ACTAGT 5---A CTAGT---3SpeI Sphaerotilus natans 3TGATCA 3---TGATC A---5 Streptomyces 5GCATGC 5---GCATG C---3SphI[48] phaeochromogenes 3CGTACG 3---C GTACG---5 Streptomyces 5AGGCCT 5---AGG CCT---3StuI[49][50] tubercidicus 3TCCGGA 3---TCC GGA---5 5TCTAGA 5---T CTAGA---3 53XbaI[48] Xanthomonas badrii
  54. 54. Enzyme Source Recognition Sequence Cut 5---G AATTC--- 5GAATTC 3EcoRI Escherichia coli 3CTTAAG 3---CTTAA G--- 5 5CCWGG 5--- CCWGG---3EcoRII Escherichia coli 3GGWCC 3---GGWCC ---5 5---G GATCC--- 5GGATCC 3BamHI Bacillus amyloliquefaciens 3CCTAGG 3---CCTAG G--- 5 5TCGA 5---T CGA---3TaqI Thermus aquaticus 3AGCT 3---AGC T---5 5GANTCA 5---G ANTC---3HinfI Haemophilus influenzae 3CTNAGT 3---CTNA G---5 5GATC 5--- GATC---3Sau3A Staphylococcus aureus 3CTAG 3---CTAG ---5 5CAGCTG 5---CAG CTG---3PovII* Proteus vulgaris 3GTCGAC 3---GTC GAC---5 5CCCGGG 5---CCC GGG---3SmaI* Serratia marcescens 3GGGCCC 3---GGG CCC---5 54
  55. 55. APPLICATIONS: They are used to assist insertion of genes into plasmid vectors during gene cloning and protein expression experiment. Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms . 55
  56. 56. Restriction enzyme can be used to genotype a DNA sample without the need for expensive gene sequencing. Restriction enzymes are used to digest genomic DNA for gene analysis by Southern blot. 56
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