1. 1

2. DNA SEQUENCING, MODIFICATION & RESTRICTION 2 3. 3 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. 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. 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. 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 8. 8 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. 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. 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 nucleotide11 12. 12 13. 13 14. 14 15. Reading a DNA Sequencing Gel Sequence 5 to 3 C G G G C G T 15 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 fragments16 17. MAXAM-GILBERT SEQUENCINGThis 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. 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. ChemicalChemicalBaseChemical used for used for used forspecificitybase alterationaltered basestrandremovalcleavageG Dimethylsulphate PiperidinePiperidineA+G Acid AcidPiperidineC+THydrazine PiperidinePiperidineHydrazine + HighCPiperidinePiperidinesaltA>CAlkaliPiperidinePiperidine19 20. G A+G C +T C SequenceCG T TCC G G A C T A A20 21. Automated DNA Sequencing with Fluorescent DyesEach different ddNTP is coupled to a different coloredfluorescent dyeddTTP is red; ddGTP is black etc.21 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. 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 TNucleotide added : A T C A G C T 23 24. Bisulfite Sequencing Bisulfite sequencing is used to detect methylation inDNA. Bisulfite deaminates cytosine, making uracil. Methylated cytosine is not changed by bisulfitetreatment. The bisulfite-treated template is then sequenced.24 25. Bisulfite Sequencing The sequence of treated and untreated templates iscompared. Me MeMe Methylated sequence:GTCGGCGATCTATCGTGCA Me MeMe Treated sequence: GTCGGCGATUTATCGTGUA DNA Sequence: (Untreated) reference: ...GTCGGCGATCTATCGTGCA Treated sequence:...GTCGGCGATUTATCGTGUAThis sequence indicates that these Cs are methylated. 25 26. Genome sequencing strategies Only short DNA molecules (~800 bp) can be sequencedin one read, so large DNA molecules, such asgenomes, longer sequences must be subdivided intosmaller fragments and subsequently reassembled to givethe overall sequence. Genome sequencing can be approached in two ways26 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. Clone contig sequencing: Involves the systematic production andsequencing of sub clones arrange overlappingclones before sequencing. 28 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. MODIFICATION 30 31. MODIFICATION It is the enzymatic alteration of its own DNA by thecell, in a species distinctive way , thus differentiating itfrom that of other species. The protective modification of the host cell DNA isbrought about by modification methylases,whichmethylate certain adenine residues. Once the host cell DNA is modified in this manner,itcannot be degraded by that cells restriction enzymes. 31 32. The restriction methylases transfer methyl groupsfrom s-adenosylmethionine to pairs of adenineresidues in duplex DNA , one in each strand; the twoadenine are on adjacent or near by base pairs. The sequence of bases on the two stands between andnear the methylated adenines is symmetrical on eitherside of a mid point.32 33. DNA RESTRICTION33 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 35. 35 36. Restriction Enzymes scan theDNA sequence. 36 37. recognition site 5-GTATAC-3 :::::: 3-CATATG-5 A palindromic recognition site reads the same on thereverse strand as it does on the forward strand when bothare read in the same orientation. Restriction enzymes recognize a specific sequence ofnucleotides and produce a double-stranded cut in theDNA. There are two types of palindromic sequences that can bepossible in DNA.37 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 greaterbiological importance than mirror-like palindromes. 38 39. Different restriction enzymes that recognize the samesequence are known as neoschizomers. These often cleave in different locales of the sequence.Different enzymes that recognize and cleave in the samelocation are known as isoschizomers. Types Restriction endonucleases are categorized into three orfour general groups (Types I, II and III) based on theircomposition and enzyme cofactor requirements, the natureof their target sequence, and the position of their DNAcleavage site relative to the target sequence. 39 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 requirements40 41. Type I restriction enzymes were the first to be identifiedand were first identified in two different strains (K-12 andB) of E. coli. These enzymes cut at a site that differs, and is a randomdistance (at least 1000 bp) away, from their recognitionsite. Cleavage at these random sites follows a process of DNAtranslocation, which shows that these enzymes are alsomolecular motors. The recognition site is asymmetrical and is composed oftwo specific portionsone containing 34nucleotides, and another containing 45 nucleotidesseparated by a non-specific spacer of about 68nucleotides. 41 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. 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. Type II: 44 45. They are a dimer of only one type of subunit; their recognition sites are usually undivided and palindromic and 48 nucleotides in length, they recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activitythey usually require only Mg2+ as a cofactor.[ These are the most commonly available and used restriction enzymes.45 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. Type IIB restriction enzymes (e.g. BcgI and BplI) aremultimers, containing more than one subunit They cleave DNA on both sides of their recognition to cutout the recognition site. They require both AdoMet and Mg2+ cofactors. Type IIErestriction endonucleases (e.g. NaeI) cleave DNAfollowing interaction with two copies of their recognitionsequence.[ One recognition site acts as the target for cleavage, whilethe other acts as an allosteric effector that speeds up orimproves the efficiency of enzyme cleavage. 47 48. Type IIG restriction endonucleases (Eco57I) do have asingle subunit, like classical Type II restrictionenzymes, but require the cofactor AdoMet to be active. Type IIM restriction endonucleases, such as DpnI, are ableto recognize and cut methylated DNA Type IIS restriction endonucleases (e.g. FokI) cleave DNAat a defined distance from their non-palindromicasymmetric recognition sites These enzymes may function as dimers. Similarly, Type IITrestriction enzymes (e.g., Bpu10I and BslI) are composed oftwo different subunits 48 49. Type III restriction enzymes (e.g. EcoP15) recognize twoseparate non-palindromic sequences that are inverselyoriented. They cut DNA about 20-30 base pairs after therecognition site. These enzymes contain more than one subunit and requireAdoMet and ATP cofactors for their roles in DNAmethylation and restriction, respectively. They are components of prokaryotic DNA restriction-modification mechanisms that protect the organismagainst invading foreign DNA.49 50. Type III enzymes are hetero-oligomeric, multifunctional proteins composed of twosubunits, Res and Mod. The Mod subunit recognises the DNA sequencespecific for the system and is a modificationmethyltransferase; as such it is functionally equivalentto the M and S subunits of type I restrictionendonuclease. Res is required for restriction, although it has noenzymatic activity on its own. 50 51. Type III enzymes recognise short 5-6 bp longasymmetric DNA sequences and cleave 25-27 bpdownstream to leave short, single-stranded 5protrusions They require the presence of two inversely orientedunmethylated recognition sites for restriction to occur.These enzymes methylate only one strand of theDNA, at the N-6 position of adenosyl residues, sonewly replicated DNA will have only one strandmethylated, which is sufficient to protect againstrestriction. 51 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. 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 Streptomyces5GAGCTC 5---GAGCT C---3SacI[48] achromogenes3CTCGAG 3---C TCGAG---5 5GTCGAC 5---G TCGAC---3SalI[48] Streptomyces albus 3CAGCTG 3---CAGCT G---5 Streptomyces5AGTACT 5---AGT ACT---3ScaI[48] caespitosus 3TCATGA 3---TCA TGA---5 5ACTAGT 5---A CTAGT---3SpeI Sphaerotilus natans 3TGATCA 3---TGATC A---5 Streptomyces5GCATGC 5---GCATG C---3SphI[48] phaeochromogenes3CGTACG 3---C GTACG---5 Streptomyces5AGGCCT 5---AGG CCT---3StuI[49][50] tubercidicus3TCCGGA 3---TCC GGA---5 5TCTAGA 5---T CTAGA---3 53XbaI[48] Xanthomonas badrii 54. EnzymeSourceRecognition SequenceCut5---G AATTC--- 5GAATTC 3EcoRI Escherichia coli 3CTTAAG 3---CTTAA G---5 5CCWGG5--- CCWGG---3EcoRIIEscherichia coli 3GGWCC3---GGWCC ---55---G GATCC--- 5GGATCC 3BamHI Bacillus amyloliquefaciens 3CCTAGG 3---CCTAG G---5 5TCGA 5---T CGA---3TaqIThermus 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---CAGCTG---3PovII*Proteus vulgaris 3GTCGAC 3---GTCGAC---5 5CCCGGG 5---CCCGGG---3SmaI* Serratia marcescens 3GGGCCC 3---GGGCCC---5 54 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. 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 57. 57