1. Section J
Analysis of cloned DNA

2. J1 Characterization of clones
J2 acid sequencing Nucleic
J3 Organization of cloned genes
J4 Mutagenesis of cloned genes

3. J1 Characterization of clones
J1-2 Restriction mapping
J1-3 Partial digestion
J1-4 Southern and Northern blotting

4. J1-1 Characterization
Preparation of pure DNA is the first step of any characterization
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.
Restriction digestion & agarose gel electrophoresis using molecular weight marker

5. J1-2 Restriction Mapping
Cleavage pattern of the insert DNA by restriction enzymes. Useful in determining the order of multiple fragments (genes).
 HindIII
BamHI
EcoRI /BamHI
EcoRI
8259
6580
4840
4539
3419
2860
1740
1120
EcoRI
BamHI
4840
1740
1120
3419
Target DNA
pAMP
EcoRI
BamHI
1120
3419

7. * * * * J1-3 Partial digestion 6 kb 4 kb 3 kb
10 kb insert *
6 kb *
4 kb *
3 kb
End-labeled radioactive DNA
10 kb
6 kb
partial digestion
4 kb
3 kb
Agarose electrophoresis
autoradiography

8. J1-4 Southern and Northern blotting
DNA on blot
RNA on blot
Genomic DNA preparation RNA preparation
Restriction digestion
Denature with alkali
Agarose gel electrophoresis 
DNA blotting/transfer and fixation RNA
6. Probe labeling 
6. Hybridization (temperature) 
7. Signal detection (X-ray film or antibody) 

9. Southern analysis 酶切 电泳 转膜 显影 杂交

10. Northern analysis
How?

11. J2 Nucleic acid sequencing
J2-1 DNA sequencing
J2-2 RNA sequencing
J2-3 Sequence databases
J2-4 Genome sequencing projects

12. 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.

13. Sanger’s enzymic method
ddA
AddT
ATddC
ATCddG
ATCGddC
ATCGCddG
M13
primer
Klenow fragment
dATP dTTP
dGTP dCTP
dideoxy dATP T
思考：为什么ddNTP使DNA合成终止？

14. Automatic
sequencer
Fluorescence
Labeled ddNTP
2. Polymerase catalyzed

15. Maxam and Gilbert
chemical method

16. J2-2 RNA sequencing RNase T1 （G） RNase U2 （A）
RNase Phy M （A+U） Bacillus cereus RNase （U+C）

17. J2-3 Sequence databases Genebank ; EMBL 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.

20. EMBL

21. 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。

22. J2-4 Genome sequencing projects
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.
Human genome project

23. 思考：为什么两种载体不同？ ---GGCATGACTCTCTGCCTA--- YAC 400,000bp EcoRI BamHI
PLASMID
COSMID
40,000bp
4,000bp
思考：为什么两种载体不同？
首先必须进行overlapping 分析，以确定YAC之间的关系。
给插入不同cosmid内的序列排序,需要配合STS, RFLPs等其他技术.
STSs sequence-tagged sites
在人类,目标是获得30000个STSs(间隔100kb)作为人类基因组序列的landmarker.网上有STSs database.
cosmid clone 也用于学者间的交换和散布.

24. 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

25. J3-1 Organiztion 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.

26. J3-2 Mapping cDNA on Genomic DNA
Genomic DNA clone
DNA
RNA
S1 nuclease
cDNA clone
思考：碱的作用是什么？
with electron microscopy to determine if the gene contains introns，and the number and the length of introns。
with gel electrophsis to verify the number and the length of introns。
Alkali
ss DNA fragments
( Number and length )

27. J3-3 S1 nuclease mapping S1 nuclease 活性特点？ M13 150bp Genomic DNA gene
400bp Sau 3A
gene
Genomic DNA
150bp
Start point for
transcription
PAGE
DNA
Anneal
Sau 3A
M13
Alkali
DNA
mRNA
mRNA
Digestion with S1 nuclease will now produce a protected fragment。 one end of which is marked by a restriction site， the other by the transcription unit teminus or the exon-intron boundary or start point。
the goal is to find out the 3- or 5- terminus of transcript in the genomic DNA。
S1 nuclease
活性特点？
determines the precise 5’- and 3’- ends of RNA transcripts. Sequence ladder is required to determine the precise position

28. J3-4 Primer extension 思考：与有何Southern blot联系？ Genomic DNA  gel
cDNA fragments  probes
Restriction maps E
3kb
1kb
2kb
4kb
cDNA
DNA
total
/partial
/auto
3kb
1kb
2kb
4kb
10kb
6kb
4kb
3kb
1。找出基因组DNA和cDNA各酶切片端间的关系。
2。使用一端标记的cDNA作为探针，确定基因组DNA中内含子顺序，即基因极性。
3。同样办法可用于制备基因图谱。尤其适用愚酶切片段多而复杂的情形。
思考：与有何Southern blot联系？

29. the regulatory protein
J3-5 Gel retardation
gene
Mix with
the regulatory protein
control 2 1 3 4 5 1 2 3 4 5
DNA binding protein
1、to search for regulatory sequences，with “real regulatory protein”or its crude extract。
2、with labeled 5- or 3- 、exonuclease and gel electrophsis，it is possible to determine the precise sequence for protein binding。
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.

30. J3-6 DNase I footprinting
活性特点？
PAGE
DNase I
Regulatory protein
DNase I produce one cleavage per molecule。

31. 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.
Reporter gene
promoter
enhancers
基因前的特定序列
Control element
可以配合缺失验证功能。
-galactosidase
CAT
luciferase

32. J4 Mutagenesis of cloned genes
J4-1 Deletion mutagenesis
J4-2 Site-directed mutagenesis
J4-3 PCR mutagenesis

33. J4-1 Deletion 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 .

34. Exonuclease III
S1 or mung bean nuclease
Ligation

35. J4-2 Site-directed mutagenesis
Formerly,single-stranded templates prepared using M13 were used,but now PCR techniques are now preferred.

36. 思考：引物设计的要点？ Primer 1 Primer 2 TTC AAG Primer 3 Primer 4 First PCR A
Remove primer Denature, anneal
second PCR
思考：引物设计的要点？