質體 DNA 以內限制酶 切割並以電泳分析

What are Restriction endonucleases? Enzymes that attack and digest internal regions of the DNA. First enzyme extracted from E. coli (cut randomly and not always close to the desired site).

Types of Restriction endonucleases Type I cuts at random sites. Type III cuts at specific sites quite near the recognition sequence, but may be difficult to predict. ATP required for source of energy.

Types of Restriction endonucleases Type II restriction enzymes: ◦ 1) Each cuts in a predictable and consistent manner at a site within or adjacent to the recognition sequence. ◦ 2) ATP not needed. Only a cofactor Mg++ is needed. ◦ 3) More than 1200 type II have been isolated from a variety of prokaryotic organisms ◦ 4) More than 100 types are commercially available

Property of restriction enzymes They break the phosphodiester bonds that link adjacent nucleotides in DNA molecules.

Nomenclature of Restriction endonucleases 1) First letter: initial letter of the genus name of the organism from which the enzyme is isolated. 2) Second and third letter: usually initial letters of the organism’s species name.

Nomenclature of Restriction endonucleases 3) Fourth letter (if any): indicates a particular strain of organism 4) Roman numerals: originally indicate the order in which enzymes from the same organism and strain are eluted from chromatography column. More often, though, indicate the order of discovery.

Examples of Type II restriction enzymes EcoRIE = genus Escherichia co = species coli R = strain RY13 I= first endonuclease isolated

Examples of Type II restriction enzymes BamHI B = genus Bacilus am = species amyloliquefaciens H = strain H I = first endonuclease isolated

Examples of Type II restriction enzymes HindIII H = genus Haemophilus in = species influenzae d = strain Rd III = third endonuclease isolated

Most type 2 RE recognize and cleave DNA within particular sequences of 4 to 8 nucleotides which have two fold axis of rotational symmetry. Such sequences are often referred as palindromes: Ex: HaeIII 5’ TGACGGGTTCGA GGCC AG 3’ 3’ ACTGCCCAAGGT CCGG TC 5’

Restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites. (sticky end) (blunt end)

13 一般而言 recognition site 越長，則此 site 出現在 DNA 上的機率也就越小 (sticky end) (blunt end) EcoRI GAATTC CTTAAG ScaI AGTACT TCATGA Recognition size of enzyme 出現機率 TetramerSau3AIGATC1/4 4 =1/256 PentamerEcoRIICC(A/T)GG1/4 5 =1/1024 HexamerEcoRIGAATTC1/4 6 =1/4096 OctomerNotIGCGGCCGC1/4 8 Palindromic sequences

Ends of restriction fragments; Blunt ends Sticky ends ◦ 3‘ extensions ◦ 5‘ extensions Importantly, the 5' termini of each strand in the cleavage product(s) retain the phosphoryl group from the phosphodiester bond, the 3' termini are hydroxylated.

Blunt ends Some restriction enzymes cut DNA at opposite base They leave blunt ended DNA fragments AluI HaeIII

Sticky ends Most restriction enzymes make staggered cuts Staggered cuts produce single stranded “sticky-ends”

19 Closed circular DNA Open circular DNA

General uses of REs Detection of RFLPs Restriction enzyme map: The location of the restriction enzyme cleavage sites on the DNA molecule DNA fragments from different species can be ligated to create Recombinant DNA:

取 10 ul 質體 DNA (pET-32a) 分別加入 1ul 之 Xho 1 與 Xba I 於 37 ℃水浴槽中作用 1 小時 ①限制酶切割 加入 13 ul ddH 2 O 加入 5ul NEB buffer 實驗流程 NEB buffer ： 提供酵素產生最佳反應之緩衝液

③跑膠 吸取 10ul 混和樣品 緩慢 loading 至 well 中 ( 記取 loading 位置 ) 加入 3ul DNA marker ( 助教執行 ) 設定 100 伏特電壓， 40min ②樣品製備 各取 10ul 酵素切割後與未切 割之樣品與 2ul loading dye 進行跑膠 ( 均勻混和 ) 將膠體浸入含有 Ethidium bromide (EtBr) 之染劑 ( 約 10~15 秒鐘 ) 取出後，以 dH 2 O 稍微清洗 單手戴手套 ( 只可觸碰膠體 嚴禁觸摸任何東西 ) 置於 UV 照相裝置中 調節膠體影像並照相 ④染膠

Xba I enzyme site (729) Xho I enzyme site (158) 5'...C↓T C G A G...3' 3'...G A G C T↑C...5' 5'...T↓C T A G A...3' 3'...A G A T C↑T...5'

未切 Xho I/ Xba I Xho IXba I 5K 6K 4K 3K 2K 1K 經限制酶切割前後之質體 DNA 電泳圖

-6K- -5K- -4K- -7K- 雙切雙切 Marker 雙切雙切 未切未切 未切未切