Restriction Enzyme Digestion Of Vector Or Dna Insert For Subcloning

Selection of Restriction Enzymes

Restriction enzymes or endonucleases recognize specific base sequences in DNA and make a cut on both strands. In general, these enzymes can be divided into two types. Type A refers to endonucleases that can produce sticky ends after digestion. Type B enzymes generate blunt ends following digestion. Hundreds of restriction enzymes are commercially available. Typical type A enzymes include BamH I, Hind III, EcoR I, Kpn I, Not I, Sac I, Xho I and Xba I. They can recognize and produce sticky ends as follows.

The common type B enzymes that generate blunt ends are as follows:

Dra i:







-- 3







-- 5

EcoR V:







-- 3







-- 5

Sma i:







-- 3







-- 5

Tips: (1) Selection of restriction enzymes depends on the particular restriction enzyme cutting site in the vectors and on the DNA to be inserted. To make the best choice, it is necessary to find out which enzyme has a unique cut at the right place by using a computer program such as GCG or Eugene (see Chapter 6). In our experience, several selections can be made. Choice A: two incompatible, sticky ends in the vector and the same sticky ends in the DNA insert will be the best choice for high efficiency of ligation and transformation. For example, for the DNA insert to have Xho I and Hind III sites at both ends of the DNA to be subcloned, and if the vector also has unique Xho I and Hind III sites at the multiple cloning site, is very fortunate. The vector and insert DNA can be easily digested with these two enzymes. After purification of the digested DNA, vectors or inserts cannot undergo self-ligation because of the two incompatible sticky ends produced by these two enzymes. During ligation, only insert and vector DNA can be combined. In theory, any colonies after antibiotic selection will be positive transformants. If all goes well, putative constructs can be obtained in a few days starting from restriction enzyme digestion to verification of positive colonies. (2) Choice B: if two unique restriction enzyme sites cannot be found at the right place, one unique site will be acceptable. For instance, if each of the vectors and inserts has a unique EcoR I site at the right site for cloning, both vector and insert can then be digested with EcoR I. The disadvantage is that, compared with choice A, choice B will have less efficiency of ligation between vector and insert and lower efficiency of transformation. Because the same single sticky end is present in both vector and DNA insert, the vector or insert will undergo a high percentage of self-ligation even though the vectors are treated with alkaline phosphatase. As a result, most of the colonies selected by antibiotics may be from self-ligation vectors. This will be time consuming and costly to verify the selected colonies. Nonetheless, single, sticky-end ligation will allow simultaneously making sense and antisense orientations of the DNA insert. In other words, the DNA of interest will be inserted into the vector in both directions, with a probability of 1:1. This is particularly useful if it is desirable to make both sense and antisense constructs of cDNA to be expressed. (3) Choice C: f a unique enzyme site available at the right place is a blunt-end enzyme, it may be necessary to use a blunt-end ligation approach. Compared with choices A and B, choice C has the lowest efficiency of ligation and transformation. It is known that blunt-end ligation is tough. A very small percentage of digested vectors and inserts can be ligated together at 4 or 16°C. Even worse, a significant portion of the ligated DNA molecules is self-ligated DNA. It usually takes weeks or months to obtain putative DNA constructs. Obviously, this is not effective way to clone and may cause frustration.

Selection of Cloning Vectors

Commercial plasmids such as pcDNA3, pcDNA 3.1, pMAMneo, pGEM series and pBluescript-SK II are available for subcloning. Selection of particular vectors depends on individual investigators. In general, a standard plasmid for subcloning of a DNA fragment should contain the following necessary fragments: (1) a poly-cloning site for the insertion of the DNA of interest, (2) SP6, T7, or T3, or equivalent promoters upstream from the multiple cloning site (MCS) in opposite directions in order to express the DNA insert for sense RNA, or antisense RNA, or for protein analysis, (3) the origin of replication for the duplication of the recombinant plasmid in the host cells, and (4) a selectable marker gene such as Ampr for antibiotic selection of transformants.

Protocols for Restriction Enzyme Digestion

1. For single-restriction enzyme digestion, set up the following reaction on ice:

DNA (vector or insert DNA), 10 to 12 mg in 6 ml 10X Appropriate restriction enzyme buffer, 2.5 ml Appropriate restriction enzyme, 3.3 units/mg DNA Add dd.H2O to a final volume of 25 ml.

The following reaction is designed for double-restriction enzyme digestion: DNA (vector or insert DNA), 10 to 12 mg 6 ml 10X Appropriate restriction enzyme buffer, 3 ml Appropriate restriction enzyme A, 3 units/mg DNA Appropriate restriction enzyme B, 3 units/mg DNA Add dd.H2O to a final volume of 30 ml.

Notes: (1) The restriction enzymes used for vector and insert DNA digestions should be the same in order to ensure optimal ligation. The digestions should be carried out in separate tubes for the vector and insert DNA. (2) For double-restriction enzyme digestions, the appropriate 10X buffer containing a higher NaCl concentration than the other buffer may be used for the double-enzyme digestion. (3) If two restriction enzymes require different reaction buffers, it is better to set up two consecutive single-restriction digestions to ensure the DNA will be completely cut. Specifically, perform one enzyme digestion of the DNA and precipitate the DNA afterwards. The DNA can be dissolved in dd.H2O and carry out the second enzyme digestion.

2. Incubate the reaction at an appropriate temperature (e.g., 37°C) for 2 h. For single-enzyme digested DNA, proceed to step 3. For double-enzyme digested DNA, proceed to step 5.

Tips: (1) To obtain an optimal ligation, the vector and insert DNA should be completely digested. The digestion efficiency may be checked by loading 1 fig of the digested DNA with loading buffer in a 1% agarose minigel. Meanwhile, the undigested vector and insert DNA (1 fig) and standard DNA markers should be loaded into the adjacent wells. After electrophoresis, the undigested plasmid DNA may display multiple bands because of different levels of supercoiled plasmids. However, one band should be visible for a complete, single-enzyme digestion; one major band and one tiny band (<70 bp) may be visible after digestion with two different restriction enzyme digestions of the vectors. (2) After completion of restriction enzyme digestion, calf intestinal alkaline phosphatase (CIAP) treatment should be carried out for single-restriction enzyme digestion of vectors. This treatment serves to remove 5'-phosphate groups, thus preventing recircularization of the vector during ligation. Otherwise, the efficiency of ligation between the vector and insert DNA will be very low. For vectors digested with double-restriction enzymes, the CIAP treatment is not necessary.

3. Carry out the CIAP treatment as follows. 10X CIAP buffer, 5 fl Single-enzyme digested DNA, 25 fl

CIAP diluted in 10X CIAP buffer, 0.01 unit/pmol ends Add dd.H2O to a final volume of 50 fl.

Notes: (1) CIAP and 10X CIAP should be kept at 4°C. (2) Calculate pmol ends; for example, 9 fg digested DNA is left after using 1 fg of 10 fg digested DNA for analysis in an agarose gel. If the vector size is 3.2 kb, the pmol ends can be calculated by the formula below:

amount of DNA

base pairs x 660/pair

4. Incubate the reaction at 37°C for 1 h and add 2 ml of 0.5 M EDTA buffer (pH 8.0) to terminate the reaction. At this point, one of two options can be chosen. If the vector or insert DNA will be a single fragment following restriction enzyme, the digested DNA can be purified by protein extraction and ethanol precipitation as described next. However, if restriction enzyme digestion results in more than one fragment or band, the fragment of interest should be purified by agarose gel electrophoresis as described in the next section.

5. Add one volume of TE-saturated phenol/chloroform to the reaction. Mix well by vortexing the tube for 1 min and centrifuge at 11,000 x g for 5 min at room temperature.

6. Carefully transfer the top, aqueous phase to a fresh tube and add one volume of chloroform:isoamyl alcohol (24:1) to the supernatant to further extract proteins. Mix well and centrifuge as in step 5.

7. Carefully transfer the upper, aqueous phase to a fresh tube and add 0.1 volume of 3 M sodium acetate buffer (pH 5.2) or 0.5 volume of 7.5 M ammonium acetate to the supernatant for efficient precipitation. Briefly mix and add 2 to 2.5 volumes of chilled 100% ethanol to the supernatant and precipitate the DNA at -80°C for 1 h or at -20°C for 2 h.

8. Centrifuge at 12,000 x g for 10 min and carefully decant the supernatant. Add 1 ml of 70% ethanol to the tube and centrifuge at 12,000 x g for 5 min. Dry the DNA pellet under vacuum for 20 min. Dissolve the DNA in 10 ml dd.H2O. Take 1 ml of the sample to measure the concentration of the DNA at A260 nm. Store the sample at -20°C prior to use. Proceed to DNA ligation.

Reagents Needed

Appropriate enzymes

10X Appropriate restriction enzyme buffer

1% Agarose minigel

TE-saturated phenol/chloroform

Chloroform:isoamyl alcohol (24:1)

7.5 M Ammonium acetate

3 M Sodium acetate buffer, pH 5.2

Calf intestinal alkaline phosphates (CIAP)

TE buffer

10X CIAP Buffer

0.5 M Tris-HCl, pH 9.0 1 mM ZnCl2 10 mM MgCl2 10 mM Spermidine

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