Analysis of cloned DNASection J

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)

Southern analysis酶切

电泳

转膜

杂交 显影

Northern analysis

How?

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？

Automatic sequencer

1. Fluorescence Labeled ddNTP

2. Polymerase catalyzed

Maxam and Gilbert chemical method

RNase T1 （ G ） RNase U2 （ A ）RNase Phy M （ A+U ） Bacillus cereus RNase （ U+C ）

J2-2 RNA sequencing

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

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

DNase IPAGE

J3-6 DNase I footprinting

Regulatory protein 活性特点？

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

Exonuclease III

S1 or mung bean nuclease

Ligation

J4-2 Site-directed mutagenesis

Formerly,single-stranded templates prepared using M13 were used,but now PCR techniques are now preferred.

Primer 1

Primer 2

TTC

AAGPrimer 3

Primer 4

First PCR

A

TTCAAG

TTCAAG

Remove primer Denature, anneal

TTCAAG

Primer 1

Primer 2

second PCR

TTCAAG

思考：引物设计的要点？