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6 copies of GAATTC: it will be cut into

7 fragments which could be separated in the gel electrophoresis by size (The largest fragment) (The smallest fragment) digestionof a DNA fragment with endonuclease Eco RI ? Endonucleases are used to make restriction map: C e.g. the combination of EcoRI + HindIII C Allows different regions of one molecule to be isolate and a given molecule to be identified C A given molecule will generate a characteristic series of patterns when digested with a set of different enzymes Restriction digestion (1) Restriction enzymes differ in the recognition specificity recognition specificity: target sites are different. (2) Restriction enzymes differ in the the length they recognized length they recognized, and thus the frequencies differ. (3) Restriction enzymes differ in the the nature of the DNA ends they generate nature of the DNA ends they generate: blunt/flush ends (平末端), sticky/staggered ends (粘性末端). (4) Restriction enzymes differ in the cleavage activity cleavage activity. sticky ends blunt ends recognition sequences and cut sites of various endonucleases Restriction digestion ? The 5'

protruding ends are said to be sticky because they readily anneal through base-pairing to DNA molecules cut with the same enzyme Reanneal with its complementary strand or other strands with the same cut Restriction digestion RFLP (Restriction fragment length polymorphism, 限制性片段长度多态性) ? 由于DNA多态性,取代的碱基正好位于 某一限制酶切割识别顺序,那么不同个体, 不同染色体上的DNA用相同酶切割时产 生不同长度的DNA片段,这种现象叫 RFLP. ? 等位片段数一般是2―3个,即有酶切点和 无酶切点. RLFP分析法 4.Identification by Hybridization (杂交 鉴定) DNA hybridization can be used DNA hybridization can be used to identify specific DNA to identify specific DNA molecules molecules Nucleic acid DNA hybridization Hybridization: the process of base-pairing between complementary ssDNA or RNA from two different sources. A labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence. Probe (探针) The mixture being probed has typically either been separated by size on a gel, or is distributed as a library in different colonies DNA hybridization Labeling of DNA or RNA probes End labeling: put the labels at the ends Uniform labeling: put the labels internally radioactive labeling: display and/or magnify the signals by radioactivity Non-radioactive labeling: display and/or magnify the signals by antigen labeling C antibody binding C enzyme binding - substrate application (signal release)-fluorescent labeling DNA hybridization End labeling Single stranded DNA/RNA 5'

-end labeling: polynucleotide kinase (PNK) 3'

-end labeling: terminal transferase DNA hybridization DNA hybridization How to label one end of a DNA: Labeling at both ends by kinase, then remove one end by restriction digestion. G CTTAAp5'

5'

pAATTC G DNA hybridization Uniformly labeling of DNA/RNA Nick translation labeling of DNA: DNase I to introduce random nicks into template DNA ? DNA pol I to remove dNMPs from 3'

to 5'

and add new dNMP including labeled nucleotide at the 3'

ends. Hexanucleotide primered labeling of DNA: Denature DNA ? add random hexanucleotide primers and DNA pol ? synthesis of new strand incorporating labeled nucleotide. DNA hybridization Nick Translation Nick Translation OH p OH+ P P DNA dNTP ,DNA酶I, DNA聚合酶I 32P-dNTP Bio-dNTP DNA hybridization 随机引物标记探针 随机引物标记探针 5` 3` 5` 5` 3` DNA 32p-dNTP,Bio-Dntp 6bp primer Klenow,dNTP 变性-复姓 DNA hybridization Strand-specific DNA probes: e.g. M13 DNA as template the missing strand can be re- synthesized by incorporating radioactive nulceotides Strand-specific RNA probes: labeled by in vitro transcription of the desired RNA sequence. DNA hybridization Hybridization probes can Hybridization probes can identify electrophoretically identify electrophoretically- - separated DNAs and RNAs separated DNAs and RNAs DNA hybridization