Restriction enzymes 5 overhangs

Biolabs (Pickering, ON), and Promega (Madison, WI). It is important to note that restriction enzymes producing 3 -overhangs should be avoided if possible (e.g., Psfl, Sfil, Kpnl). The use of such enzymes has been reported to result in the production of additional, nonspecific transcripts (Schenborn and Mierendorf, 1985). If these enzymes must be used, an exonuclease such as DNA Polymerase 1 Large (Klenow) Fragment can be utilized to convert the overhang to a blunt end before the template is transcribed. 

By contrast, other restriction enzymes cut at different portions of a DNA molecule, forming an "offset" break with "sticky" ends. For example, the restriction enzyme known as BamHI cuts between two GG nitrogen bases, but at different parts of the DNA molecule, forming "overhangs or sticky ends. These sticky ends provide locations at which new nitrogen base sequences can be inserted. 

If you are using the restriction enzyme BseR I or another enzyme that produces overhangs, do not alter the DNA concentration in this step, because activity of the mung bean nuclease that is added directly to this reaction is concentration dependent. 

Sticky ends. After digestion of a DNA with certain restriction enzymes, the ends left have one strand overhanging the other to form a short (typically 4 nt) single-stranded segment. This overhang will easily re-attach to other ends like it and become known as sticky ends. ... 

Figure 7 Cloning by restriction digest and ligation. Both the PCR-amplified gene of interest and the target vector are digested with the same restriction enzymes (or with enzymes that produce compatible ends). The short section of overhanging sequence (called sticky ends ) are complementary to each other. The digested DNAs are mixed, treated with ligase, and transformed into a suitable Escherichia coli host strain to produce a new recombinant DNA molecule with the gene of interest specifically inserted into a host vector.

The 3 -ends are labeled, after specific restriction enzymes have produced a protruding 5 -end, by a fill-in reaction that allows only one label to be introduced (e.g., labeling with dATP with an overhang with 2 Ts should be avoided). Reverse transcriptase or Sequenase are the preferred enzymes for this fill-in although the Klenow fragment has been widely used. The 3 -> 5 exonuclease activity of Klenow, however, may remove the protruding template end. 

EXAMPLE 8.17 Among the most important enzymes of recombinant DNA technology are the restriction enzymes. These are endonucleases that cleave DNA only at specific sequences of bases (called restriction sites). Typically, restriction sites are palindromic, in other words, the sequences are the same in the 5 — 3 and 3 — 5 strands. Restriction enzymes are produced by bacteria as an antiviral defense, and they cleave the DNA of viruses (bacteriophages) that infect them. However, they do not cleave host bacterial DNA. Fig. 8-17 shows the restriction sites of three common restriction enzymes, BamHI, EcoRI, and PvuII. Because BamHI and EcoRI cleave their restriction site asymmetrically, they produce overhangs in the cleaved DNA, called sticky ends. Conversely, PvuII cleaves symmetrically, producing blunt ends. 

Extraneous transcripts have been reported to occur during the preparation of the riboprobe when the template contains 3 protruding ends therefore, do not use restriction enzymes that generate 3 overhangs. If there is no alternative restriction site, the 3 overhang should be converted to a blunt end using Klenow DNA polymerase. 

---