Section J – Analysis and uses of cloned DNA

1. Section J – Analysis and uses of cloned DNA

2. Plant cell Animal cell

3. Contents J1 Characterization of clones Characterization, Restriction mapping, Partial digestion, Labeling nucleic acid, Southern and Northern blotting J2 Nucleic acid sequencing DNA sequencing, RNA sequencing, Sequence databases, Analysis of sequences, Genome sequencing projects J3 Polymerase of cloned genes PCR, the PCR cycle, Template, Primers, Enzymes, PCR optimization, PCR variations J4 Organization of cloned genes Organization, Mapping cDNA on genomic DNA, S1 nuclease mapping, Primer extension, Gel retardation, DNaseⅠ footprinting, Reporter genes J5 Mutagenesis of cloned genes Deletion mutagenesis, Site-directed mutagenesis, PCR mutagenesis J6 Applications of cloned genes Applications, Recombinant protein, Genetically modified organisms, DNA fingerprinting, Medical diagnosis, Gene therapy

4. J1 Characterization of clones — Characterization Preparation of pure DNA is the first step of any characterization. Plasmid DNA: from bacterial colonies Bacteiophage DNA: Plaque purified phage → → infecting a bacterial culture → → cell lysis → → phage particles → → phenol-chloroform, ethanol precipitate → → Bacteiophage DNA

5. J1 Characterization of clones — Restriction mapping • The most common application of restriction mapping is presented: Determining the orientation of a cloned insert. This method requires that restriction maps of the cloning vector and the insert are already available. Example1：

6. J1 Characterization of clones — Restriction mapping Example2：

7. J1 Characterization of clones — Partial digestion 2kb 4kb 3kb 1kb 10 kb insert Complete digestion Partial digestion 10 kb 7 kb 6 kb 4 kb Can not delineate the restriction sites. 3 kb 2 kb 1 kb

8. Delineate the restriction sites by partial digested end-labeled radioactive DNA. E E E 3kb 1kb 2kb 4kb * 10 kb insert * 6 kb * 4 kb 3 kb * 10 kb End-labeled radioactive DNA 6 kb 4 kb Partial digestion 3 kb Agarose electrophoresis Autoradiography

9. J1 Characterization of clones — Labeling nucleic acid Radioactive labeling: display and/or magnify the signals by radioactivity Non-radioactive labeling: display and/or magnify the signals by Biotin and digoxin etc 1.End labeling: put the labels at the ends 2.Uniform labeling: put the labels internally

10. End labeling • （1）Single stranded DNA/RNA 5’-end labeling:dephosphorylation  polynucleotide kinase 3’-end labeling: terminal transferase

11. （2）Double stranded DNA/RNA Fill in the recessive 3’-ends（ 3’-凹端） by DNA polymerase. Labeled at both ends ---------------------G ---------------------CTTAAp5’ 5’pAATTC G For restriction mapping, cut the DNA with another enzyme

12. 2. Uniformly labeling of DNA/RNA （1）Nick translation （切口平移）: DNase I to introduce random nicks DNA Pol I to remove dNTPs from 5’ to 3’ and add new dNTP including labeled nucleotide at the 3’ ends.

13. （2）Hexanucleotide primed labeling(六聚核苷酸引物标记，random labeling 随机标记）: Denature DNA  add random hexanucleotide primers and DNA pol  synthesis of new strand incorporating labeled nucleotide.

14. 3. Specific probes （1）Strand-specific DNA probes: e.g.M13 DNA as template the missing strand can be re- synthesized by incorporating radioactive nucleotides.

15. （2）Strand-specific RNA probes

16. J1 Characterization of clones — Southern and Northern blotting • Southern blotting, for detecting DNA ; • Northern blotting, for detecting RNA; • Western blotting, for detecting protein.

17. 1.Genomic DNA preparation 2.Restriction digestion 3.Denature with alkali 4.Agarose gel electrophoresis 5.DNA blotting/ transfer and fixation 6.Probe labeling 7.Hybridization (temperature) 8.Signal detection (X-ray film or antibody)

18. Southern analysis

19. Northern blotting

21. J2 Nucleic acid sequencing — DNA sequencing • Three main methods: 1. Maxam and Gilbert chemical method  2. Sanger`s enzymatic method  3. Sequencing by hybridization (SBH)

22. 1. Maxam and Gilbert chemical method • The end-labeled DNA is subjected to base-specific cleavage reactions prior to gel separation. • Modification of bases: • Methylation by dimethyl sulfate ：G(DMS) • Formic acid: PurinesA & G • Hydrazine : hydrolyzeT & C • Hydrazine + high salt: onlyC

23. A A A G A T T A A G C C * Dimethyl sulfate Formic acid Hydrazine Hydrazine+high salt G A+G C+T C 烷基转移酶

24. 2. Sanger`s enzymatic method Uses dideoxynucleotides as chain terminators to produce a ladder of molecules generated by polymerase extension of primer

25. G C T A Sanger’s method Template +primer (15-17nt) +dNTPs +ddNTPs +[35S]dATP +T7 DNA pol PAGE Autoradiography 3’GTGACTACTCAGGCACTTGCTTTGCC5’

27. Automatic sequencer

28. 3. Sequencing by hybridization(SBH)

29. J2 Nucleic acid sequencing — RNA sequencing • By base-specific cleavage of5’-end-labeledRNA using RNases that cleave 3’ to a particular nucleotide.Partial digestionis required to generatea ladderof cleavage products which are analyzed byPAGE.

30. RNase T1: cleaves after G RNase U2: after A RNase Phy M: after A and U Bacillus cereus RNase: after U and C

31. J2 Nucleic acid sequencing — Sequence databases • DDBJ(日本国家遗传学研究所)http://www.ddbj.nig.ac.jp • EMBL-EBI (欧洲生物信息研究所) :http://www.ebi.ac.uk/Databases/index.html • Genbank at NCBI （美国国家生物技术信息中心）: http://www.ncbi.nlm.nih.gov

35. J2 Nucleic acid sequencing — Analysis of sequences • Using computers and software packages, such as GCG sequence analysis package. • 1. Identify important sequence features such as restriction sites, open reading frames, start and stop codons, as well as potential promoter sites, intron-exon junctions, etc.

36. ORF #2 ORF #1 100 200 300 400 500 600 700 Sequence analysis of a cloned DNA sequence revealed some important features

37. 2. Homology search by BLAST (NCBI) or FASTA (EBI): Compare new sequence with all other known sequences in the databases, which can determine whether related sequences have been obtained before.

38. J2 Nucleic acid sequencing — Genome sequencing projects • With the development ofautomated DNA sequencersandrobotic workstations to prepare samples for sequencing, the entire genome sequence of several organisms have been determined. • Many phages and viruses • Several Bacteria (E. coli, 4 x 106) • Plant (Arabidopsis 6.4 x 107 , rice) • Human 3.3 x 109

39. J3 Polymerase of cloned genes — PCR • The polymerase chain reaction (PCR)： • To amplify a sequence of DNA using a pair of primers each complementary to one end of the DNA target sequence.

40. J3 Polymerase of cloned genes — the PCR cycle • Denaturation :The target DNA (template) is separated into two stands by heating to95℃ • Primer annealing :The temperature is reduced to around55℃to allow theprimersto anneal. • Polymerization (elongation, extension):The temperature is increased to 72℃ for optimal polymerization step which uses updNTPsand required Mg++.

43. J3 Polymerase of cloned genes — Template • Single-or double-stranded form; • The size of the template DNA is not critical; • In the case of mammalian or plant genomic DNA, up to 1.0 ug of DNA is utilized per reaction. The typical amounts of yeast, bacterial, and plasmid DNAs used per reaction are 10 ng, 1ng, and 1pg, respectively; • Template DNA is dissoved in 10 mM Tris-Cl (pH 7.6) containing a low concentration of EDTA (<0.1 mM).

44. J3 Polymerase of cloned genes — Primers • PCR primers：about18 to 30nt long and with similar G+C contents. • Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can beTm-5oC.

45. If the target DNA is not known,there is only limited amino acid sequence available. Degenerate primers An oligo pool derived from protein sequence. E.g. His-Phe-Pro-Phe-Met-Lys can generate a primer CAU(CAC)-UUU(UUC)-CCU(CCC,CCA,CCG)- UUU(UUC)-AUG-AAA(AAG) 2x2x4x2x2 =64

46. J3 Polymerase of cloned genes — Enzymes • The most common isTaq polymerasefrom Thermus aquaticus. It has no 3’ to 5’ proofreading exonuclease activity. Accuracy is low, not good for cloning. • Pfu(Pyrococcus furiosus, Promega & Stratagene),

47. J3 Polymerase of cloned genes — PCR optimization • PCR cycle • Enzymes • Template DNA • Mg++

48. J3 Polymerase of cloned genes — PCR variations 1. Inverse PCR, IPCR 2. Anchored PCR, APCR 3. asym metric PCR 4. Reverse transcription RT-PCR 5. 修饰引物PCR 6. Nest PCR 7. multiplex PCR 8. 重组PCR 9. differential PCR, d-PCR 10. quantitative PCR, qPCR 11. in situ PCR 12. immuno-PCR 13. Thermal Asymmetric Interlaced PCR，TAIL-PCR

49. J4 Organization of cloned genes — Organization • The 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 transcription • regulatory sequences.

50. J4 Organization of cloned genes — Mapping cDNA on genomic DNA • The genomic clone is digested on a gel and then subjected to Southern blot using all or part of the cDNA as a probe. Show which genomic restriction fragments contain cDNAsequences Using a probe from one end of a cDNA can show thepolarity of the genein the genomic clone. Some of the restriction sites will be common in both clones but may be different distances apart.