分子生物学教学课件：4-Section J Analysis and uses of cloned DNA

1、Section JAnalysis of cloned DNAJ1, J2, J3 Major TechniquesJ4 Organization of cloned genesJ5 Mutagenesis of cloned genes Restriction mappingSequencing (DNA & RNA)Northern and Southern blottingPolymerase chain reaction (PCR)Major Techniques usedThis Techniques may be used for other purpose as well

2、J1. Characterization of clonesDetermining 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 characterizationSi

3、ze of DNA fragment clonedRestriction digestion & agarose gel electrophoresis using molecular weight markerinsert0.8 kb0.5 kb1.0 kb1.6 kb2.0 kb3.0 kb4.0 kb3.5 kbRestriction Mapping 限制性酶切图谱限制性酶切图谱Cleavage pattern of the insert DNA by restriction enzymes. Useful in determining the order of multiple

4、 fragments (genes).1. Combinational enzyme digestion2. Partial digestion （部分消解）（部分消解）1. Combinational enzyme digestionNonessential regionLong (left)armshort (right)arm phageSal I: 19 kb, 15 kb, 9 kb HindIII: 21 kb, 11 kb, 7 kb, 4 kbSalI + HindIII: 19 kb, 7 kb, 6 kb, 5 kb, 4 kb, 2 kb19 kb9 kb15 kb21

5、kb11 kb7 kb4 kbHindIIISal IS + HDouble digestionNonessential regionLong (left)armshort (right)armSS19 kb9 kb15 kb21 kbH4 kb11 kb7 kbHHDelineate the restriction sites on the DNA 10 kb insert*End-labeled radioactive DNApartial digestionAgarose electrophoresisautoradiography3 kb4 kb6 kb10 kb3 kb4 kb6 k

6、bDelineate the restriction sites by partial digested end-labeled radioactive DNA EEEJ2 Nucleic acid sequencingn DNA sequencingn RNA sequencingn Sequence databasesn Analysis of sequencesn Genome sequencing projectsDNA sequencingTwo main methods:nMaxam and Gilbert chemical method the end-labeled DNA i

7、s subjected to base-specific cleavage reactions prior to gel separation.nSangers enzymatic method ( ) the latter uses dideoxynucleotidesdideoxynucleotides as chain terminators to produce a ladder of molecules generated by polymerase extension of primer. Maxam and Gilbert chemical method Modification

8、 of bases:G: methylation by dimethyl sulfate (DMS)Purines A & G: formic acidHydrazine (联氨联氨): hydrolyze T & CHydrazine + high salt: only CCleavage after modified bases: piperidine (氮杂环己烷）氮杂环己烷）chain-termination methodnddNTPs are chain-terminating nucleotides: the synthesis of a DNA strand st

9、ops when a ddNTP is added to the 3 endThe absence of 3-hydroxyl lead to the inefficiency of the nucleophilic attack on the next incoming substrate moleculeDNA synthesis aborts at a frequency of 1/100 every time the polymerase meets a ddGTP Tell from the gel the position of each GA C G T3GTGACTACTCAG

10、GCACTTGCTTTGCC5Sangers method:Template+primer (15-17nt)+dNTPs+ddNTPs+35SdATP+T7 DNA polPAGEAutoradiographyFig 20-15 DNA sequencing gel4 systems with dNTP+ ddGTP, dNTP+ ddATP d NTP+ ddCTP, d NTP+ ddTTP separately“read” the sequencing gel to get the sequence of the DNAAutomatic sequencer1.Fluorescence

11、 Labeled ddNTP2. Polymerase catalyzed The shortgun strategy permits a partial assembly of large genome sequencenIf we want to sequence a much larger and more complicate eukaryotic genome using the shortgun strategy. What can we do?nFirstly, libraries in different level should be constructed.Fig 20-1

12、6nThe DNA fragment can be easily extracted and sequenced automatically.nSophisticated computer programs have been developed to assemble the randomized DNA fragment, forming contigs.nA single contig is about 50000 to 200000 bp. Its useful to analysis fruit fly genome that contains an average of one g

13、ene every 10kb.nIf we want to analysis human genome, contigs should be assembled into scaffolds. 1-16 the paired-end strategy permits the assembly of large genome sequencenThe main limitation to producing large contigs is the occurrence of repetitive sequence. (Why?)nTo solve this problem, paired-en

14、d sequencing is developed.nThe same genomic DNA is also used to produce recombinant libraries composed of large fragments between 3100kb.NUCLEIC ACIDSnThe end of each clone can be sequenced easily, and these larger clones can firstly assemble together. nIf a larger scaffold is needed, you should use

15、 a cloning vector that can carry large DNA fragment, (at least 100kb). BAC is a good choice. BAC(bacterial artificial chromosome): a special cloning vector to obtain paired-end sequence data from large DAN fragments that are at least 100kb in length.The use of BACs often permits the assignment of mu

16、ltiple contigs into a single scaffold of several megabases. genome-wide analysisnThe purpose of this analysis is to predict the coding sequence and other functional sequence in the genome.nFor the genomes of bacteria and simple eukaryotes, finding ORF is very simple and effective.NUCLEIC ACIDSGene f

17、inder methods:analysis of protein-coding regions in cionanThe most important method for validating protein coding regions and identify those those missed by current current gene finder program is the use of cDNA sequence data.nThe mRNAs are firstly reverse transcript into cDNA, and these cDNA, both

18、full length and partial, are sequenced using shortgun method. These sequence are used to generate EST (expressed sequence tag) database. And these ESTs are aligned onto genomic scaffolds to help us identify genes.Comparative Genome Analysis Comparative analysis helps identify short exons, some locat

19、ed near the 5end of the gene and the core promoter, that are often missed by gene prediction programs.One of the striking findings of comparative genome analysis is the high degree of synteny, conservation in genetic linkage, between distantly related animals.Synteny in the mouse and human chromosom

20、esThe availability of whole genome sequences for an increasing number of animals is providing a rapidly expanding database for comparative genomics. The exon-intron nature of eukaryotic genes and the lack of strict sequence constraints in noncoding elements create formidable challenges to the identi

21、fication of protein-coding sequences and regulatory elements by computational approaches. New and more effective tools of bioinformatics will be required to fully epxploit the treasure trove of information that is being generated by automated DAN sequencing.Analysis of sequencesnUsing computers and

22、software packages, such as GCG sequence analysis package offered by Univ. of Wisconsin1.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.100100200200300300400400500500600600700700ORF #1ORF #2Sequence analysis of a clo