Presentation on theme: "Section J Analysis of cloned DNA"— Presentation transcript:
1 Section J Analysis of cloned DNA Molecular Biology CourseSection JAnalysis of cloned DNA
2 J1 Characterization of clones J2 Nucleic acid sequencing Molecular Biology CourseJ1 Characterization of clonesJ2 Nucleic acid sequencingJ3 Polymerase chain reactionJ4 Organization of cloned genesJ5 Mutagenesis of cloned genesJ6 Application of cloning
3 Major Techniques used Restriction mapping Sequencing (DNA & RNA) Analysis of cloned DNA- overviewMajor Techniques usedRestriction mappingSequencing (DNA & RNA)Northern and Southern blottingPCRThese Techniques may be used for other purpose as well
4 J1 Characterization of clones Analysis of cloned DNAJ1 Characterization of clonesJ1-1 CharacterizationJ1-2 Restriction mappingJ2-3 Partial digestionJ2-4 Labeling nucleic acidJ2-5 Southern and Northern blotting
5 J1 Characterization of clones Determining various properties of a recombinant DNA molecule, such as size, restriction map, nucleotide sequence, whether containing a gene (transcribed sequence), the position and polarity of any gene.Preparation of pure DNA is the first step of any characterization
6 Size of DNA fragment cloned J1 Characterization of clonesSize of DNA fragment clonedRestriction digestion & agarose gel electrophoresis using molecular weight marker4.0 kb3.5 kb3.0 kb2.0 kb1.6 kb1.0 kbinsert0.8 kb0.5 kb
7 J1-2 Restriction Mapping J1 Characterization of clonesJ1-2 Restriction MappingCleavage pattern of the insert DNA by restriction enzymes. Useful in determining the order of multiple fragments (genes).1. Combinational enzyme digestion2. Partial digestion
11 Delineate the restriction sites by partial digested end-labeled radioactive DNA *10 kb insert*6 kb*4 kb*3 kbEnd-labeled radioactive DNA10 kb6 kbpartial digestion4 kb3 kbAgarose electrophoresisautoradiography
12 End labeling: put the labels at the ends J1 Characterization of clonesJ1-4 Labeling of DNA or RNA probesradioactive labeling: display and/or magnify the signals by radioactivityNon-radioactive labeling: display and/or magnify the signals by antigen labeling – antibody binding – enzyme binding - substrate application (signal releaseEnd labeling: put the labels at the endsUniform labeling: put the labels internally
13 Single stranded DNA/RNA J1 Characterization of clonesEnd labelingSingle stranded DNA/RNA5’-end labeling: dephosphorylation polynucleotide kinase (PNK)3’-end labeling: terminal transferase
15 Double stranded DNA/RNA J1 Characterization of clonesEnd labelingDouble stranded DNA/RNAFill in the recessive 3’-ends by DNA polymeraseLabeled at both ends5’pAATTCGGCTTAAp5’For restriction mapping, cut the DNA with another enzyme
16 Uniformly labeling of DNA/RNA Nick translation: J1 Characterization of clonesUniformly labeling of DNA/RNANick translation:DNase I to introduce random nicks DNA polI to remove dNMPs from 3’ to 5’ and add new dNMP including labeled nucleotide at the 3’ ends.Hexanucleotide primered labeling:Denature DNA add random hexanucleotide primers and DNA pol synthesis of new strand incorporating labeled nucleotide .
17 Strand-specific DNA probes: e.g.M13 DNA as template J1 Characterization of clonesStrand-specific DNA probes:e.g.M13 DNA as templatethe missing strand can be re- synthesized by incorporating radioactive nulceotidesStrand-specific RNA probes:labeled by transcription
18 J1-5 Southern and Northern blotting J1 Characterization of clonesJ1-5 Southern and Northern blottingDNA on blotRNA on blotGenomic DNA preparation RNA preparationRestriction digestionDenature with alkaliAgarose gel electrophoresis DNA blotting/transfer and fixation RNA6. Probe labeling 6. Hybridization (temperature) 7. Signal detection (X-ray film or antibody)
19 J1 Characterization of clones Southern analysis
20 J1 Characterization of clones Steps of Southern blot
21 Northern analysis COB RNAs in S. cerevisiae bI1bI2bI3bI4bI5mRNAPre-mRNAs
22 Blot type Target Probe Applications J1 Characterization of clonesBlot typeTargetProbeApplicationsSouthernDNADNA or RNAmapping genomic clones estimating gene numbersNorthernRNARNA sizes, abundance, and expressionWesternProteinAntibodiesprotein size, abundance
23 J2 Nucleic acid sequencing Analysis and uses of cloned DNAJ2 Nucleic acid sequencingJ2-1 DNA sequencingJ2-2 RNA sequencingJ2-3 Sequence databasesJ2-4 Analysis of sequencesJ2-5 Genome sequencing projects
24 J2-1 DNA sequencing J2 nucleic acid sequencing Two main methods: Maxam and Gilbert chemical methodthe end-labeled DNA is subjected to base-specific cleavage reactions prior to gel separation.Sanger`s enzymic method ()the latter uses dideoxynucleotides as chain terminators to produce a ladder of molecules generated by polymerase extension of primer.
26 J2 nucleic acid sequencing Maxam and Gilbertchemical methodDNA labeled at oneend with 32PGCTGCTABase modificationGCTGCTACH3Release or displace-ment of reacted basesGCTCTAStrand scissionGCTCTA
27 J2 nucleic acid sequencing 32pGpCpTpGpCpTpApGpGpTpGpCpCpGpApGpCChain cleavageat guanines32p32pGpCpTp32pGpCpTpGpCpTpApMaxam-Gilbert sequencing. We methylate guanines with a mild DMS treatment that methylates on average one guanine per DNA strand.Then use piperidine to remove the methylated base and break the DNA strand at the apurinic site.32pGpCpTpGpCpTpApGp32pGpCpTpGpCpTpApGpGpTp32pGpCpTpGpCpTpApGpGpTpGpCpCp32pGpCpTpGpCpTpApGpGpTpGpCpCpGpAp32pGpCpTpGpCpTpApGpGpTpGpCpCpGpApGpC
28 Sanger sequencing J2 nucleic acid sequencing This figure shows the structure of a dideoxynucleotide (notice the H atom attached to the 3' carbon). Also depicted in this figure are the ingredients for a Sanger reaction. Notice the different lengths of labeled strands produced in this reaction.
29 J2 nucleic acid sequencing This figure is a representation of an acrylamide sequencing gel. Notice that the sequence of the strand of DNA complementary to the sequenced strand is 5' to 3' ACGCCCGAGTAGCCCAGATT while the sequence of the sequenced strand, 5' to 3', is AATCTGGGCTACTCGGGCGT.
32 J2 nucleic acid sequencing RNA sequencingIt is sometimes necessary to sequence RNA directly, especially to determine the position of modified nucleotides present in, eg, tRNA and rRNA.This is achieved by base-specific cleavage of 5’-end-labeled RNA using RNases (ribonuclease) that cleave 3’ to a particular nucleotide. Partial digestion is required to generate a ladder of cleavage products which are analyzed by PAGE.
33 RNase T1: cleaves after G RNase U2: after A RNase Phy M: after A and U J2 nucleic acid sequencingRNase T1: cleaves after GRNase U2: after ARNase Phy M: after A and UBacillus cereus RNase: after U and C
34 T1 cleaved 0 2 10 20 50 G 43 P 9 P8 P3’ P7 P 6 P5 P4 J3/4 P 2.1 P 2 642595121/122157222308P 2P 2.1J3/4P4P5P 6111P3’P7P8P 9T1 cleaved
35 J2 nucleic acid sequencing J2-3 Sequence databasesTwo largest DNA databases of are EMBL in Europe and Genbank in the USA.Newly determined DNA,RNA and protein sequence are entered into databases.The collections of all known sequences are available for analysis by computer.
38 J2-4 Analysis of sequences J2 nucleic acid sequencingJ2-4 Analysis of sequencesUsing computers and software packages, such as GCG sequence analysis package offered by Univ. of WisconsinIdentify important sequence features such as restriction sites,open reading frames,start and stop codons, as well as potential promoter sites, intron-exon junctions,etc.
39 J2 nucleic acid sequencing ORF #2ORF #1100200300400500600700Sequence analysis of a cloned DNA sequence revealed some important features
40 J2 nucleic acid sequencing 2. compare new sequence with all other known sequences in the databases, which can determine whether related sequences have been obtained before.
41 J2-5 Genome sequencing projects J2 nucleic acid sequencingJ2-5 Genome sequencing projectsWith the development of automated DNA sequencers and robotic workstations to prepare samples for sequencing,the entire genome sequence of several organisms have been determined.Many phages and virusesSeveral Bacteria (E. coli, 4 x 106)Plant (Arabidopsis 6.4 x 107 , rice)Human 3 x 109
42 J3 Polymerase chain reaction Analysis and uses of cloned DNAJ3 Polymerase chain reactionJ3-1 PCRJ3-2 The PCR cycleJ3-3 TemplateJ3-4 PrimersJ3-5 EnzymesJ3-6 PCR optimization
43 J3 Polymerase chain reaction J3-1 PCRThe polymerase chain reaction(PCR) is to used to amplify a sequence of DNA using a pair of primers each complementary to one end of the the DNA target sequence.
44 J3 Polymerase chain reaction J3-2 The PCR cycleDenaturation: The target DNA (template) is separated into two stands by heating to 95℃Primer annealing: The temperature is reduced to around 55℃ to allow the primers to anneal.Polymerization (elongation, extension): The temperature is increased to 72℃ for optimal polymerization step which uses up dNTPs and required Mg++.
46 J2 nucleic acid sequencing Fig.Steps of PCRTemplatePrimersEnzymes
47 J3 Polymerase chain reaction J3-3 TemplateAny source of DNA that provides one or more target molecules can in principle be used as a template for PCRWhatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed.
48 J3 Polymerase chain reaction J3-4 PrimersPCR primers need to be about 18 to 30 nt long and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures.They are designed to anneal on opposite strands of the target sequence.Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is nt, annealing temperature can be Tm5oC
49 Degenerate primers: an oligo pool derived from protein sequence. J3 Polymerase chain reactionDegenerate primers: an oligo pool derived from protein sequence.E.g. His-Phe-Pro-Phe-Met-Lys can generate a primer5’-CAY TTY CCN TTY ATG AARY= PyrimidineN= any baseR= purine
50 J3-5&6 Enzymes and PCR Optimization J3 Polymerase chain reactionJ3-5&6 Enzymes and PCR OptimizationThe most common is Taq polymerase.It has no 3’ to 5’ proofreading exonuclease activity. Accuracy is low, not good for cloning.We can change the annealing temperature and the Mg++ concentration or carry out nested PCR to optimize PCR.
54 J4 Organiztaion of cloned genes Analysis and uses of cloned DNAJ4 Organiztaion of cloned genesJ4-1 OrganizationJ4-2Mapping cDNA on Genomic DNA (where)J4-3 S1 nuclease mapping (5’ and 3’ end)J4-4 Primer extension (5’ end)J4-5 Gel retardation (binding protein)J4-6 DNase I footprinting (protein binding sites)J4-7 Reporter genes (promoter study)
55 J4-1 Organiztion J4 Organization of cloned genes cDNA clones have defined organization.A run of A residues defines the clone’s 3’-end.There will be a stop codon at its upstream. If the clone is complete, there also will be a start condon. These two codon indicates an ORF.
56 It can be determined by mapping and probing experiments J4 Organization of cloned genesThe presence and polarity of any gene in a genomic clone is not obvious (5’ and 3’ end)It can be determined by mapping and probing experimentsTo determine:which genomic sequences are present in the mature mRNA transcriptThe absent sequences are usually introns and sequences upstream of the transcription start site and down stream of the 3’-processing site.Start and stop sites for transcriptionregulatory sequences.
57 J4-2 Mapping cDNA on genomic DNA J4 Organization of cloned genesJ4-2 Mapping cDNA on genomic DNAThe genomic clone is digested on a gel and then subjected to Southern blot using all or part of the cDNA as a probe.Using full length cDNA as probe can show which genomic restriction fragments contain sequences also present in the cDNAUsing a probe from one end of a cDNA can show the polarity of the gene in the genomic clone.Some of the restriction sites will be common in both clones but may be different distances apart. These can often help to determine the organization of the genomic clone.
58 J4 Organization of cloned genes J4-3 S1 nuclease mapping determines the precise 5’- and 3’- ends of RNA transcripts. Sequence ladder is required to determine the precise positionS1 nuclease is an enzyme which specifically hydrolyses single-stranded RNA or DNA.RNA 5’3’*DNA 3’5’Add S1 nucleaseRNA 5’3’5’DNA 3’PAGE Analysis
59 J4-4 Primer extension J4 Organization of cloned genes Determine the 5’ ends of RNA molecules using reverse transcriptase to extend an antisense DNA primer in the 5’ to 3’ direction. Sequence ladder is required to determine the precise position
60 J4 Organization of cloned genes J4-5 Gel retardationMixing a protein extract with a labeled DNA fragment and running the mixture on a native gel will show the presence of DNA-protein complex as retarded bands on the gel.Protein bound withDNA/RNALabeled freeDNA/RNA
61 J4 Organization of cloned genes DNA bound totwo proteinsDNA-proteincomplexBare DNAFig Gel retardation A short labeled nucleic acid is mixed with a cell or nuclear extract expected to contain the binding protein. Then, samples of labeled nucleic acid, with and without extract, are run on a gel. The DNA-protein complexes are shown by the presence of slowly migrating bands.
62 J4-6 Dnase I footprinting J4 Organization of cloned genesJ4-6 Dnase I footprintingIdentify the actual region of sequence with which the protein interacts.Sequence ladder is required to determine the precise positionAATAAG*5’
63 J4 Organization of cloned genes Bind proteinFig DNase footprinting The protein protects DNA from attack by DNase. We treat the DNA -protein complex with DNase I under mild conditions, so that an average of only one cut occur per DNA molecule.DNase(mild),then removeprotein and denature DNAElectrophoresis,autoradiograph
64 J4 Organization of cloned genes ProteinConc:TCGA15The three lanes represent DNA that was bound to 0, 1, and 5 units of protein. The lane with no protein shows a regular ladder of fragments. The lane with one unit shows some protection, and the lane with 5 units shows complete protection in the middle. We usually include sequencing reactions performed on the same DNA in parallel lanes, which tells exactly where the protein bound.
65 J4 Organization of cloned genes J4-7 Reporter genesTo study the function of a control element of a gene (promoter and regulatory elements), reporter genes such as b-galactosidase to “report” the promoter action.
66 J5 Mutagenesis of cloned genes Analysis and uses of cloned DNAJ5 Mutagenesis of cloned genesJ5-1 Deletion mutagenesisJ5-2 Site-directed mutagenesisJ5-3 PCR mutagenesis
67 J5-1 Deletion mutagenesis J5 Mutagenesis of cloned genesJ5-1 Deletion mutagenesisIn the cDNA clones,it is common to delete progressively from the ends of the coding region to discover with parts of the whole protein have properties.In genomic clones,when the transcription part has been identified,upstream are removed progressively to discover the minimum length of upstream sequence that has promoter and regulatory function .
68 J5 Mutagenesis of cloned genes Exonuclease IIIS1 or mung bean nucleaseLigation
69 J5-2 Site-directed mutagenesis J5 Mutagenesis of cloned genesJ5-2 Site-directed mutagenesisFormerly,single-stranded templates prepared using M13 were used,but now PCR techniques are now preferred.
70 J5-3 PCR mutagenesis Deletion or point mutation J5 Mutagenesis of cloned genesJ5-3 PCR mutagenesisDeletion or point mutation
71 J5 Mutagenesis of cloned genes Forward mutagenic primerSP6 primerT7 primerReverse mutagenic primerFirst PCRPCR mutagenesis Two separate PCR reactions are performed, one amplifying the 5’-portion of the insert using SP6 and the reverse primer, and the other amplifying the 3’-portion of the insert using the forward and T7 primers.Remove primersDenature and anneal
72 J5 Mutagenesis of cloned genes Extend and dosecond PCRSP6 primerT7 primerPCR mutagenesis Two separate PCR reactions are performed, one amplifying the 5’-portion of the insert using SP6 and the reverse primer, and the other amplifying the 3’-portion of the insert using the forward and T7 primers.Subclone
74 P(deltaP5abc) construction J5 Mutagenesis of cloned genesPCRP(deltaP5abc) constructionE1-P5P5’-E2elongationPCRP5abcexonintron
75 J6 Applications of cloning Analysis of cloned DNAJ6 Applications of cloningJ6-1 ApplicationsJ6-2 Recombinant proteinJ6-3Genetically modified organismsJ6-4 DNA fingerprintingJ6-5 Medical diagnosisJ6-5 Gene therapy
76 J6-1 Applications J6 Applications of cloning CLONING Recombinant GeneticallyModifiedOrganismsDNAfingerprintingCLONINGRecombinantproteinGenetherapyMedicaldiagnosis
77 J6-2 Recombinant protein J6 Applications of cloningJ6-2 Recombinant protein·Prior to the advent of gene cloning, production of protein was to purify them from tissues. Drawbacks: small amounts, viral contamination etc. ·Gene cloning has circumvented the listed problems.
79 J6 Applications of cloning Prokaryotic expression system can be used to produce eukaryotic proteins, but there are some problems:Only cDNA clones can be used asthey contain no intronsInsoluble, precipitatedFusion proteinLack of eukaryotic post-translational modifications
80 J6 Applications of cloning These problems can be solved by using the eukaryotic expression systems, such as the yeast, Baculovirus and humn cell lines.
81 J6-3 Genetically modified organisms J6 Applications of cloningJ6-3 Genetically modified organismsGenetically modified organisms(GMOs) are created when cloned genes are introduced into germ cells.In eukaryotes, if the introduced genes are derived from another organism, the resulting transgenic plants or animals can be propagated by normal breeding.e.g. A tomato has a gene for a ripening enzyme inactivated
82 J6 Applications of cloning J6-4 DNA fingerprintingHow is DNA fingerprinting done? I. Performing Southern blot II. Making a radioactive probe III.Creating a hybridization reaction IV. VNTRs
83 J6 Applications of cloning A given person's VNTRs come from the genetic information donated by his or her parents; he or she could have VNTRs inherited from his or her mother or father, or a combination, but never a VNTR either of his or her parents do not have. Shown in the left are the VNTR patterns for Mrs. Nguyen [blue], Mr. Nguyen [yellow], and their four children: D1 (the Nguyens' biological daughter), D2 (Mr. Nguyen's step-daughter, child of Mrs. Nguyen and her former husband [red]), S1 (the Nguyens' biological son), and S2 (the Nguyens' adopted son, not biologically related [his parents are light and dark green]).
84 J6 Applications of cloning The application of DNA fingerprinting: I. Paternity and maternity II.Criminal identification and forensics III.Personal identification
85 J6-5 Medical diagnosis J6 Applications of cloning A great variety of medical conditionsarise from mutation. e.g. musculardystophy, many cancers. By usingsequence information to design PCRprimers and probes, many tests havebeen developed to screen patientsfor these clinically important mutations.
86 Classical methods for scanning mutations: J6 Applications of cloningClassical methods for scanning mutations:Complete gene sequencingSingle-strand conformation analysisHeteroduplex analysisChemical cleavage of mismatch and enzymatic cleavage of mismatchProtein-truncation test
87 J6 Applications of cloning J6-6 Gene therapyAttempts have been made to treat some genetic disorders by delivering a normal copy of the defective gene to patients. This is known as gene therapy.