analysis of cloned dna section j. j1 characterization of clones j2 acid sequencing nucleic j3...
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J1 Characterization of clones
J2 acid sequencing Nucleic
J3 Organization of cloned genes
J4 Mutagenesis of cloned genes
J1 Characterization of clones
J1-1 Characterization
J1-2 Restriction mapping
J1-3 Partial digestion
J1-4 Southern and Northern blotting
Determining various properties of a recombinant DNA molecule, such as size, restriction map, nucleotide sequence, whether containing a gene, the position and polarity of any gene.
Preparation of pure DNA is the first step of any characterization
J1-1 Characterization
Restriction digestion & agarose gel electrophoresis using molecular weight marker
Cleavage pattern of the insert DNA by restriction enzymes. Useful in determining the order of multiple fragments (genes).
J1-2 Restriction Mapping
pAMP
EcoRI
BamHI1120
3419
Target DNA
EcoRI
EcoRI
BamHI
BamHI
4840
1740
1120
3419
HindIIIBamHI
EcoRI /BamHIEcoRI
825965804840453934192860
1740
1120
J1-3 Partial digestion
10 kb insert****
End-labeled radioactive DNA
partial digestion
Agarose electrophoresis
autoradiography
3 kb4 kb6 kb10 kb
3 kb4 kb
6 kb
E E E
J1-4 Southern and Northern blotting
DNA on blot RNA on blot
1.Genomic DNA preparation RNA preparation2.Restriction digestion -3.Denature with alkali - 4. Agarose gel electrophoresis 5. DNA blotting/transfer and fixation RNA6. Probe labeling 6. Hybridization (temperature) 7. Signal detection (X-ray film or antibody)
J2 Nucleic acid sequencing
J2-1 DNA sequencing
J2-2 RNA sequencing
J2-3 Sequence databases
J2-4 Genome sequencing projects
J2-1 DNA sequencing
Maxam and Gilbert chemical method
the 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.
Klenow fragmentdATP dTTPdGTP dCTPdideoxy dATP
M13
primer
T T T T T
A T G C
ddAAddTATddCATCddGATCGddCATCGCddG
Sanger’s enzymic method
思考:为什么 ddNTP 使 DNA 合成终止?思考:为什么 ddNTP 使 DNA 合成终止?
What is GenBank?GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences. There are approximately 106,533,156,756 bases in 108,431,692 sequence records in the traditional GenBank divisions and 148,165,117,763 bases in 48,443,067 sequence records in the WGS division as of August 2009.
Genebank ; EMBL
J2-3 Sequence databases
http://www.ncbi.nlm.nih.gov
EMBL ( The European Molecular Biology Laboratory ),于 1974 年由欧洲 14 个国家加上亚洲的以色列共同发起建立,现在由欧洲 30 个成员国政府支持组成,目的在于促进欧洲国家之间的合作来发展分子生物学的基础研究和改进仪器设备、教育工作等。分 7个部分:结构、分化、物理仪器、生化仪器、生物仪器、计算机和应用数学。包括一个位于德国 Heidelberg 的核心实验室,及三个位于德国 Hamburg ,法国 Grenoble 及英国Hinxton 的研究分部。由于具有开放和创新的良好学术氛围, EMBL 已发展成欧洲最重要和最核心的分子生物学基础研究和教育培训机构。目前 , 在研究中已经建立了先进的核苷酸序列数据库。
EMBL-DNA 数据库于 1982 年由 EMBL 建立,与美国的 GenBank 及日本的 DDBJ 共同组成全球性的国际 DNA 数据库,近年来发展很快,在 1995 年数据量成倍递增。 EBI 是EMBL 在英国 Hinxton 的分部,主要负责建立 EMBL-DNA 数据库,可进行核苷酸序列检索及序列相似性查询。
EMBL
• With the development of automated DNA sequencers and robotic workstations to prepare samples for sequencing,the entire genome sequence of several organisms have been determined.
J2-4 Genome sequencing projects
Human genome project
YAC 400,000bp
---GGCATGACTCTCTGCCTA---
EcoRI
BamHI
PLASMID
COSMID 40,000bp
4,000bp
思考:为什么两种载体不同?思考:为什么两种载体不同?
J3 Organiztaion of cloned genes
J3-1 Organization
J3-2 Mapping cDNA on Genomic DNA
J3-3 S1 nuclease mapping
J3-4 Primer extension
J3-5 Gel retardation
J3-6 DNase I footprinting
J3-7 Reporter genes
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.
J3-1 Organiztion
DNA
RNA
Alkali
ss DNA fragments
S1 nuclease cDNA clonecDNA clone
( Number and length )
J3-2 Mapping cDNA on Genomic DNAGenomic DNA cloneGenomic DNA clone
思考:碱的作用是什么?思考:碱的作用是什么?
150bp
Start point for transcriptionStart point for transcription
S1 nuclease
Alkali
Anneal
Sau 3A
Sau 3A
M13
PAGE
DNA
mRNA
DNA
400bp Sau 3A
geneGenomic DNAGenomic DNA
mRNA
J3-3 S1 nuclease mapping
活性特点?determines the precise 5’- and 3’- ends of RNA transcripts. Sequence ladder is required to determine the precise position
total /partial /auto
3kb
1kb
2kb
4kb
Genomic DNA gelcDNA fragments probesGenomic DNA gelcDNA fragments probes
Restriction mapsRestriction maps
10kb6kb
4kb
3kb
E E E
3kb1kb
2kb 4kb
cDNA
DNA
J3-4 Primer extension
思考:与有何 Southern blot 联系?思考:与有何 Southern blot 联系?
geneMix with
the regulatory proteincontrol
2
1
3
45
1
2
3
45
J3-5 Gel retardation
Mixing 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.
DNA binding proteinDNA binding protein
Reporter gene
promoter
enhancers
J3-7 Reporter genes
To 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.
-galactosidaseCATluciferase
基因前的特定序列Control element
J4 Mutagenesis of cloned genes
J4-1 Deletion mutagenesis
J4-2 Site-directed mutagenesis
J4-3 PCR mutagenesis
In 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 .
J4-1 Deletion mutagenesis
J4-2 Site-directed mutagenesis
Formerly,single-stranded templates prepared using M13 were used,but now PCR techniques are now preferred.