DNA Fragments are Joined by DNA Ligase

The enzyme DNA ligase is used to join DNA fragments covalently. DNA ligase operates during DNA replication where it joins up the fragments of the lagging strand (Ch.

blunt ends Ends of a double-stranded DNA molecule that are fully base paired and have no unpaired single-stranded overhang DNA ligase Enzyme that joins DNA fragments covalently, end to end sticky ends Ends of a double-stranded DNA molecule that have unpaired single-stranded overhangs, generated by a staggered cut type II restriction enzyme Type of restriction enzyme that cuts the DNA in the middle of the recognition site

FIGURE 22.03 Type II Restriction Enzymes—Blunt Versus Sticky Ends

Hpa\ is an blunt end restriction enzyme, that is, it cuts both strands of DNA in exactly the same position. EcoR\ is a sticky end restriction enzyme. The enzyme cuts between the G and A on both strands, which generates an a four base pair overhang on the ends of the DNA. Since these bases are free to base pair with any complementary sequence, they are considered "sticky".

Cut by hpa1

Blunt ends

Sticky ends ggatcc cctAgig aigat c t tctAgia

FIGURE 22.04 Matching of Compatible Sticky Ends

BamHI and Bg/II generate the same overhanging or sticky ends. BamHI recognizes the sequence 5'-GGATCC-3' and cuts after the first 5' G, which generates the 3'-CTAG-5' overhang on the bottom strand. Bg/II recognizes the sequence 5'-AGATCT-3' and cuts after the first 5' A, which generates a 5'-GATC-3' overhang on the top strand. If these two pieces are allowed to anneal, the complementary sequences will hydrogen bond together, allowing the nicks to be sealed more easily by DNA ligase.

Bam HI

g gatcc cctag g

Digest

Bgl II

a tctag gatct a

Anneal ggat c t cctaga

5). If DNA ligase finds two DNA fragments touching each other end to end, it will ligate them together (Fig. 22.05). In practice, segments of DNA with matching sticky ends will tend to stay attached much of the time and consequently DNA ligase will join them efficiently. Since DNA fragments with blunt ends have no way to bind each other, they drift apart most of the time. Ligating blunt ends is very slow and requires a high concentration of DNA ligase, as well as, a high concentration of DNA. In fact, bacterial ligase cannot join blunt ends at all. In practice, T4 ligase is normally used in genetic engineering as it can join blunt ends if need be. T4 ligase originally came from bacteriophage T4, although nowadays it is manufactured by expressing the gene that encodes it in E. coli.

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