RESTRICTION ENZYME DIGESTION OF DNA: BASIC METHOD You may be digesting your DNA just to look at it (an analytical

gel) or to cut a band out of the gel for further treatment (a preparative gel). Either way you want to be able to see the DNA bands under UV light in an ethidium-bromide-stained gel. Typically, a band is easily visible if it contains about 20ng of DNA. Now consider an example. Suppose you are digesting a plasmid that comprises 3kb of vector and 2kb of insert. You are using EcoRI (a common restriction enzyme) and you expect to see three bands: the linearised vector (3kb), the 5' end of the insert (0.5kb) and the 3' end of the insert (1.5kb). In order to see the smallest band (0.5kb) you want it to contain at least 20ng of DNA. The smallest band is 1/10th the size of the uncut plasmid. Therefore you need to cut 10x20ng, that is 200ng of DNA (0.2g). Then your three bands will contain 120ng, 20ng and 60ng of DNA respectively. All three bands will be clearly visible on the gel and the biggest band will be six times brighter than the smallest band. Now imagine cutting the same plasmid with BamHI (another popular restriction enzyme) and that BamHI only cuts the plasmid once, to linearise it. If you digest 200ng of DNA in this case then the band will contain 200ng of DNA and will be very bright and will probably be overloaded. Too much DNA loaded onto a gel is a bad thing. The band appears to run fast (implying that it is smaller than it really is) and in extreme cases can mess up the electrical field for the other bands, making them appear the wrong size also. Too little DNA is only a problem in that you will not be able to see the smallest bands because they are too faint. Having said all that, I usually digest 24L of the 50L obtained from a kit miniprep. But you see how it depends on the number and size of the bands expected. TYPICAL PROTOCOL Combine the following in a microfuge tube in order; 1l 10x Buffer 6.5l H2O 2l DNA 0.5l Enzyme Buffer: This is the 10x buffer that comes with the restriction enzyme. Most companies have about 4 different kinds of buffer (called A,B,C,D or 1,2,3,4 etc.) and occasionally a "unique buffer" for a particular enzyme. Do not worry about this. Many restriction enzymes are forgiving about buffers. EcoRI, for example, works excellently in all four of the standard buffers. You can check in the enzyme company catalogue (NEB, Stratagene, Promega, Roche etc.) where there is a page devoted to enzyme/buffer compatibility. Fundamentally it is a question of salt concentration (high or low), Magnesium concentration (high or low) and pH. H2O: Standard distilled water. It is always good to add the buffer and water into the tube first. If you put the enzyme in straight on top of the buffer then it may become irreversibly denatured DNA: As discussed above. Kit-prepared DNA is very good. If it is "home-made" miniprep DNA then salt can often be a problem. This can interfere with the enzyme (if it does not like salt) and can also make the gel run funny. Enzyme: 0.5l of enzyme is plenty for a miniprep digestion. Do not use more enzyme than 10% of the final reaction volume (ie. not more than 1l in this case). This is because the enzyme storage buffer contains antifreeze (glycerol) to allow it to survive at 20C. The glycerol will inhibit the digestion if present in sufficient quantities. Incubate for 1 hour at 37C in a waterbath. Protocol pentru dubla digestie

Enzyme EcoR1 0.5 microlitre Enzyme BamH1 0.5 microlitre buffer 1 microlitre

water 3.0 microlitre plasmid DNA 5 microlitre

Recomandri generale pentru realizarea reaciilor de digestie a unor molecule de ADN cu endonucleaze de restricie Principalele componente ale unei reacii de restricie ADN Componentele obligatorii ale oricrei reacii de restricie ADN sunt : 1. Enzima de restricie 2. ADN 3. Tamponul de reacie 4. a.d.s. (ap distilat steril) Aceste componente se adaug n urmtoarea ordine: a.d.s., tampon, ADN, enzim, toate ntr-un tub Eppi mic (0.5 ml) steril, si nu pe peretii acestuia. Se lucreaza steril, cu mnui i se schimb vrful pipetei la fiecare component al reaciei i la fiecare tub de reacie (cu excepia primului component a.d.s.) Opional: n afar de componentele obligatorii, n anumite reacii de restricie este recomandabil s se adauge: BSA (Bovine Seric Albumine) n concentraie final de 100 ug/ml (n mod special n reaciile de restricie a unor molecule mari de ADN, de ex. ADN cromozomal, sau n cazul anumitor enzime de restricie pentru care se consult cataloagele productorilor). RNazaA n concentraie final de 100-200 ug/ml (n cazul n care n etapele de purificare a ADN nu a fost fcut si tratament cu RNazaA sau n cazul RFLP pe ADN cromozomal). 1. ENZIMA 1 unitate [U] de activitate a unei enzime de restricie este definit ca fiind cantitatea de enzim necesar pentru digestia complet a 1 ug ADN substrat, n 60 min, la temperatura adecvat, folosind un tampon adecvat, ntr-un volum total de reacie de 50 ul. Aceast definiie este valabil n cazul folosirii ADN din diverse virusuri (fag, fag T4, adenovirusul Ad-2, virusul SV40) i pentru vectorii de clonare/exprimare. Cantiti de enzim recomandate : Pentru digestia unor vectori de clonare 1 5 U / ug ADN Pentru digestia unor molecule de ADN plasmidial din tulpini naturale de MO 4 8 U/ug ADN Pentru digestia unor molecule de ADN cromozomal 5 15 U / ug ADN Enzimele de restricie se stocheaz la 200C. La utilizare se menin pe ghea. n cazul majoritii enzimelor de restricie, concentraia produsului comercializat este de 10-20 U/l. Din acest motiv, n multe situaii este necesar efectuarea unei prediluii a soluiei stoc de enzim n tampon de reacie. Se recomand, de asemenea, ca volumul de enzim utilizat s reprezinte ntre 1/10i 1/5 din volumul total de reacie. Alegerea enzimei Este preferabil s se fac n funcie de ADN-ul prob, fiind necesar s se tie pentru ce enzime de restricie prezint acesta situsuri de recunoatere. Alegerea enzimelor de restricie depinde i de scopul aplicaiei: pentru restricie de ADN plasmidial se folosesc enzime obinuite (Eco RI, Hind III etc.), n timp ce pentru RFLP genomic, spre exemplu, se poate opta fie pentru enzimele uzuale (cele de mai sus), fie pentru enzime de restricie cu situsuri rare. Enzyme EcoRI Source Escherichia coli Recognition Sequence
5'GAATTC 3'CTTAAG

Cut
5'---G AATTC---3' 3'---CTTAA G---5'

EcoRII BamHI HindIII TaqI NotI HinfI Sau3A PovII* SmaI* HaeIII* HgaI[44] AluI*

Escherichia coli Bacillus amyloliquefaciens Haemophilus influenzae Thermus aquaticus Nocardia otitidis Haemophilus influenzae Staphylococcus aureus Proteus vulgaris Serratia marcescens Haemophilus aegyptius Haemophilus gallinarum Arthrobacter luteus

5'CCWGG 3'GGWCC 5'GGATCC 3'CCTAGG 5'AAGCTT 3'TTCGAA 5'TCGA 3'AGCT 5'GCGGCCGC 3'CGCCGGCG 5'GANTCA 3'CTNAGT 5'GATC 3'CTAG 5'CAGCTG 3'GTCGAC 5'CCCGGG 3'GGGCCC 5'GGCC 3'CCGG 5'GACGC 3'CTGCG 5'AGCT 3'TCGA 5'GATATC 3'CTATAG 5'CAGCAGN25NN 3'GTCGTCN25NN 5'GGTACC 3'CCATGG 5'CTGCAG 3'GACGTC 5'GAGCTC 3'CTCGAG 5'GTCGAC 3'CAGCTG 5'AGTACT 3'TCATGA 5'ACTAGT 3'TGATCA 5'GCATGC 3'CGTACG 5'AGGCCT 3'TCCGGA 5'TCTAGA 3'AGATCT

5'--CCWGG---3' 3'---GGWCC ---5' 5'---G GATCC---3' 3'---CCTAG G---5' 5'---A AGCTT---3' 3'---TTCGA A---5' 5'---T CGA---3' 3'---AGC T---5' 5'---GC GGCCGC---3' 3'---CGCCGG CG---5' 5'---G ANTC---3' 3'---CTNA G---5' 5'--GATC---3' 3'---CTAG ---5' 5'---CAG 3'---GTC 5'---CCC 3'---GGG 5'---GG 3'---CC 5'---NN 3'---NN 5'---AG 3'---TC 5'---GAT 3'---CTA CTG---3' GAC---5' GGG---3' CCC---5' CC---3' GG---5' NN---3' NN---5' CT---3' GA---5' ATC---3' TAG---5'

EcoRV* Escherichia coli EcoP15I Escherichia coli KpnI[45] PstI[45] SacI[45] SalI[45] ScaI[45] SpeI SphI[45] Klebsiella pneumoniae Providencia stuartii Streptomyces achromogenes Streptomyces albus Streptomyces caespitosus Sphaerotilus natans Streptomyces phaeochromogenes

5'---CAGCAGN25NN ---3' 3'---GTCGTCN25 NN---5' 5'---GGTAC C---3' 3'---C CATGG---5' 5'---CTGCA G---3' 3'---G ACGTC---5' 5'---GAGCT C---3' 3'---C TCGAG---5' 5'---G TCGAC---3' 3'---CAGCT G---5' 5'---AGT 3'---TCA ACT---3' TGA---5'

5'---A CTAGT---3' 3'---TGATC A---5' 5'---GCATG C---3' 3'---C GTACG---5' 5'---AGG 3'---TCC CCT---3' GGA---5'

StuI[46][47] Streptomyces tubercidicus XbaI[45] Xanthomonas badrii

5'---T CTAGA---3' 3'---AGATC T---5'

Key: * = blunt ends

N = C or G or T or A

W = A or T

2.

ADN (cantitatea de prob, puritatea si concentraia acestuia)

n reaciile de restricie, probele de ADN trebuie s fie: - suficient de pure, mai ales n ceea ce privete contaminanii proteici; - suficient de concentrate, dat fiind c se recomand ca volumul probei de ADN s nu depaeasc 1/3-1/2 din volumul total de reacie; ntr-o reacie de digestie trebuie introdus o cantitate de ADN suficient de mare pentru ca n gel s se obina fragmentele de dimensiunea dorit sau (n funcie de aplicaie) benzi vizibile. 3. Tamponul de reactie Se livreaz n preul enzimei. Este comercializat n form 10X (de zece ori concentrat), motiv pentru care se adaug n amestecul de reacie n proporie de 1/10 din volumul total al acestuia. Temperatura de reacie este de 370C, pentru majoritatea enzimelor de reacie. Trebuie verificat, ns, ntotdeauna ntr-un catalog de produse, deoarece exist i enzime de restricie care au optimum-ul de activitate la diferite alte temperaturi. Timpul de reacie Indiferent de volumul de reacie, enzima de restricie utilizat, sau varianta tehnicii de lucru aplicate timpul minim de reacie este de o or. Tipul probei ADN digerate ADN fag, vectori Plasmide naturale ADN cromozomal Timpul de reacie 1.5 - 2.5 h 3 - 5 h 6 - 24 h

Variante de stopare a reaciei de restricie - scderea temperaturii amestecului de reacie pn la 200C; - adugarea la amestecul de reacie a 0,2 volume (fa de volumul total al amestecului) de EDTANa2 0.5 M; - adugare de BFE sau BFER (n vederea ncrcrii gelului de electroforez).