Host-controlled restriction and modification system

Transcription

1 Host-controlled restriction and modification system Restriction systems allow bacteria to monitor the origin of incoming DNA and to destroy it, if it is recognized as foreign. Restriction endonucleases recognize specific sequences in the incoming DNA (e.g. phages) and cleave the DNA into fragments, either at specific sites or more randomly, thus preventing it from successfully replicating and parasitizing the cell (immunity system). The restrictive host must, of course, protect its own DNA from the potentially lethal effects of the endonuclease and so its DNA must be appropriately modified. Modification involves methylation of certain bases at a very limited number of sequences within DNA. EcoRI restriction endonuclease-methylase system (type II) Together, a restriction endonuclease and its cognate modification methyl-transferase form a restriction-modification system (R-M system)

2 Characteristics of restriction endonucleases Type I Type II Type III 1% ~ 98% < 1% Type II restriction enzymes Nearly 3000 restriction enzymes of type II have been found, exhibiting over 200 different specificities (about 200 enzymes are now purified and many of them are cloned and over-expressed in E. coli). Restriction enzymes are species non-specific: enzymes of the same specificity occur in different species (e.g BsaHI from B. stearothermophilus and AhaII from the blue-green alga Aphanothece halophytica). The genes for R.E. are often located on the chromosome, sometimes on plasmids and very occasionally located on prophages (it appears that the genes for these enzymes shuffle between microorganisms and that there is a natural selection for variety).

3 EcoRI Digestione di un frammento di DNA con l endonucleasi di restrizione EcoRI

4 Estremità piatta Estremità 5 protrudente Estremità 5 protrudente Estremità 3 protrudente Frequenza di taglio di alcuni enzimi di restrizione Sau 3A (GATC) taglio (¼)(¼)(¼)(¼) = ogni 256 bp BamHI (GGATCC) taglio (¼)(¼)(¼)(¼)(¼)(¼) = ogni ~4Kb HindII (GTPyPuAC) taglio (¼)(¼)(½)(½)(¼)(¼) = ogni ~1Kb NotI (GCGGCCGC) taglio (¼)(¼)(¼)(¼)(¼)(¼)(¼)(¼) = ogni ~ 65Kb (assumendo G/C = A/T)

5 Cutting and joining DNA molecules After a restriction endonuclease cuts DNA fragments can be joined. Features of Bacteriophage T4 DNA Ligase Polypeptide of MW= Requires ATP (the E.coli enzyme requires NAD + ). The cofactor forms an enzyme-amp complex. The complex binds to the nick and catalyzes the formation of a covalent bond in the phosphodiester chain. The optimum temperature for ligation of DNA is 37 C but at this temperature the hydrogen-bounded joint between the sticky ends is unstable (the reaction is usually carried out at 15 C). Substrate: This enzyme is active on double strand DNA with blunt ends or complementary cohesive ends that base pair to bring together 3 -OH and 5 -phosphate termini.

6 Clonaggio in un vettore plasmidico

7 DNA and RNA blotting Total RNA loaded on denaturating agarose gel 23S 16S 5S Denaturation DNA alkali treatment RNA formaldehyde gel

8 Legame del DNA o dell RNA a membrane (nitrocellulosa o nylon) e fissaggio con calore o U.V. Controllo della stringenza Specificità con cui una sonda si ibrida alla sequenza bersaglio. Una stringenza elevata si ottiene aumentando la Temp. e diminuendo la salinità (forza ionica) del tampone. Per sonde più lunghe di 100 bp: Tm = 81,5 C + 16,6 log M + 0,41 (% C+G) Per oligonucleotidi ( 20 basi): Tm = 4 C (numero di G +C) + 2 C (numero A +T)

9 Northern blotting analysis fis virf + _ virb + _ virg + _ icsb + _ cpxr + _ A Arb.Units B C

10 1) Random primer DNA labeling Denaturation (boil 5 ) Hexaprimers added Hybridization DNA synthesis with Klenow polymerase (1 hour at 37 C) and a mix of four dntps containing [α 32 P]dATP Labeled probe Denaturation (boil 5 )

11 2) Nick translation DNA degradation

12 Nonradioactive DNA probe Biotin Probe Target DNA Biotin-labeled nucleotides are incorporated into the DNA probe by random primer or nick translation methods Streptavidin Biotin-labelled alkaline phosphatase or peroxidase Substrate Light-emitting product or chromogenic substrates change color

13 End-labeling Fill-in reaction 5 -GGG 3 -CCC SmaI G-3 CTTAA-5 EcoRI dntps + [ 32 P] datp + Klenow fragment 5 -GGG GAATT-3 3 -CCC SmaI CTTAA-5 EcoRI dntps + [ 32 P] datp + Klenow fragment protruding end 3 protruding end X (PstI, SacI, KpnI, BglI) Kinase reaction DNA fragment CIP 5..pNpNpNpG 3 NpNpNpCpTpTpA 5..pNpNpNpG 3 NpNpNpCpTpTpAp Polynucleotide Kinase + [ 32 P]ATP * *

14 Sequencing by the chain-terminator or dideoxy procedure (Sanger, 1977) This method is used to sequence double stranded DNA, such as a plasmid insert or purified PCR product. The dideoxy chain termination (or enzymatic) method of DNA sequencing involves the in vitro synthesis of a DNA strand by a DNA polymerase, such as: Klenow fragment of E.coli DNA polymerase I (used in combination with cloning the DNA to be sequenced in M13 series of single-stranded vectors); Taq DNA polymerase (used in cycle sequencing); modified form of phage T7 DNA polymerase (Sequenase ). This enzyme, developed by Tabor and Richardson (P.N.A.S., 1987, vol. 84: ) is a site-directed mutant (His123 Glu) of bacteriophage gene 5 protein. Features of Sequenase: 1. unlike native enzyme can incorporate nucleotide analogs, 2. reduced exonuclease activity, 3. highly processive; catalyzing the polymerization of thousands of nucleotides without dissociating from the template.

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16 SANGER METHOD Rapid, a large n of samples can be processed simultaneously Easy to perform composition of 2-D structure of the DNA template can cause premature termination by DNA polymerase Sequencing gel

17 Primer walking La reazione di sequenza permette di stabilire con buona certezza l ordine dei primi nucleotidi. Gli inserti di DNA clonati sono solitamente molto più lunghi (5000 bp). Determinata la sequenza del primo tratto, si sintetizza un secondo primer disegnato per ibridarsi con la regione lontana circa 300 basi a valle del sito di innesco del primo primer. In maniera simile si sceglie un terzo sito legame per l innesco, si sintetizza un altro oligonucleotide e si determina la sequenza delle successive basi. La strategia Primer walking va avanti fino a completare il sequenziamento dell intero inserto.

18 Automated DNA Sequencing These systems employ fluorescent dyes attached to either the primer (I generation of this techniques) or the ddntp (II generation of this techniques). The DNA fragments produced by sequencing reactions are run through polyacrylamide gels or capillary electrophoresis. The detection systems relies on laserinduced fluorescence (helium-neon laser; 633 nm). Detecting the bands within the gel is not trivial as there are only about to moles (femtomoles) of DNA in each band. (laser)

19 A standard procedure 1) The DNA is prepared as single strand 2) A mixture of four normal (deoxy) nucleotides (dgtp, datp, dttp, dctp) 3) A mixture of four dideoxynucleotides (each present in limiting amounts) each labeled with a tag that fluoresces a different colour (ddgtp, ddatp, ddttp, ddctp) 4) DNA polymerase 5) Adequate buffer Results can be monitored in real-time on the interfaced screen and subsequently subjected to graphically interactive analysis