Recombinant DNA Technology - RESTRICTION ENZYMES CUT DNA; LIGASE JOINS DNA

 

RESTRICTION ENZYMES CUT DNA; LIGASE JOINS DNA

The ability to isolate, separate, and visualize DNA fragments would be useless unless some method was available to cut the DNA into fragments of different sizes. Luckily, naturally occurring restriction enzymes or restriction endonucleases are the key to making DNA fragments.

 

visualize 상상하다

endonucleases  ((DNA 혹은 RNA의 사슬을 분해하여 불연속화(不連續化)시키는 효소))

 

These bacterial enzymes bind to specific recognition sites on DNA and cut the backbone of both strands. They evolved to protect bacteria from foreign DNA, such as viruses. The enzymes do not cut their own cell’s DNA because they are methylation sensitive; that is, if one of the nucleotide bases in the recognition sequence is methylated, then the restriction enzyme cannot bind and therefore cannot cut the methylated DNA.

methylation 메틸기 -CH3

 

Bacteria produce modification enzymes that recognize the same sequence as the corresponding restriction enzyme. These methylate each recognition site in the bacterial genome. Therefore, the bacteria can make the restriction enzyme without endangering their own DNA.

 

 modification enzymes 변형효소

corresponding 해당하는

 

Restriction enzymes have been exploited to cut DNA at specific sites, since each restriction enzyme has a particular recognition sequence. Differences in cleavage site determine the type of restriction enzyme. Type I restriction enzymes cut the DNA strand 1000 or more base pairs from the recognition sequence.

 

exploit 이용하다

cleavage site 분열지역

 

Type II restriction enzymes cut in the middle of the recognition sequence and are the most useful for genetic engineering. Type II restriction enzymes can either cut both strands of the double helix at the same point, leaving blunt ends, or they can cut at different sites on each strand leaving single-stranded ends, sometimes called sticky ends (Fig. 3.2).

 

FIGURE 3.2 Type II Restriction Enzymes—Blunt versus Sticky Ends Hpal is a blunt-end restriction enzyme; that is, it cuts both strands of DNA in exactly the same position. EcoRI is a sticky-end restriction enzyme. The enzyme cuts between the G and A on both strands, which generates four base-pair overhangs on the ends of the DNA. Since these ends may base pair with complementary sequences, they are considered “sticky.”

 

The recognition sequences of Type II restriction enzymes are usually inverted repeats so that the enzyme cuts between the same bases on both strands. Some commonly used restriction enzymes for biotechnology applications are listed in Table 3.1. Since restriction enzymes recognize a specific nucleic sequence, these can also be used to compare the nucleotide sequence of different organisms or individuals (see Box 3.1).

 

inverted repeat(IR) 역위 반복

 

 

 

Box 3.1 Restriction Fragment Length Polymorphisms Identify Individuals
Restriction enzymes are useful for many different applications. Because the DNA sequence is different in each organism, the pattern of restriction sites will also be different. The source of isolated DNA can be identified by this pattern. If genomic DNA is isolated from one organism and cut with one particular restriction enzyme, a specific set of fragments can be separated and identified by electrophoresis. If DNA from a different organism is cut by the same restriction enzyme, a different set of fragments will be generated. This technique can be applied to DNA from two individuals from the same species. Although the DNA sequence differences will be small, restriction enzymes can be used to identify these differences. If the sequence difference falls in a restriction enzyme recognition site, it gives a restriction fragment length polymorphism (RFLP) (Fig. A). When the restriction enzyme patterns are compared, the number and size of one or two fragments will be affected for each base difference that affects a cut site.

FIGURE A [RFLP]
Analysis DNA from related organisms shows small differences in sequence that cause changes in restriction sites. In the example shown, cutting a segment of DNA from the first organism yields six fragments of different sizes (labeled a–f on the gel). If the equivalent region of DNA from a related organism were digested with the same enzyme, a similar pattern would be expected. Here, a single-nucleotide difference is present, which eliminates one of the restriction sites. Consequently, digesting this DNA produces only five fragments. Fragments c and d are no longer seen but form a new band labeled cd.

 

The number of base pairs in the recognition sequence determines the likelihood of cutting. Finding a particular sequence of four nucleotides is much more likely than finding a six basepair recognition sequence.

 

 likelihood 가능

 

So to generate fewer, longer fragments, restriction enzymes with six or more base-pair recognition sequences are used. Conversely, four base-pair enzymes give more, shorter fragments from the same original segment of DNA.

 

 

When two different DNA samples are cut with the same sticky-end restriction enzyme, all the fragments will have identical overhangs. This allows DNA fragments from two sources (e.g., two different organisms) to be linked together (Fig. 3.3).

 

identical 동일한

overhang 돌출부

 

FIGURE 3.3 Compatible Overhangs Are Linked Using DNA Ligase BamHI and Bgl Il generate the same overhanging or sticky ends: a 3′-CTAG-5′ overhang plus a 5′-GATC-3′ overhang. These are complementary and base pair by hydrogen bonding. The breaks in the DNA backbones are sealed by T4 DNA ligase, which hydrolyzes ATP to energize the reaction.

 

Fragments are linked or ligated using DNA ligase, the same enzyme that ligates the Okazaki fragments during replication (see Chapter 4). The most common ligase used is actually from T4 bacteriophage. Ligase catalyzes linkage between the 3′-OH of one strand and the 5′-PO4 of the other DNA strand. Ligase is much more efficient with overhanging sticky ends but can also link blunt ends much more slowly.

 

Restriction enzymes are naturally occurring enzymes that recognize a particular DNA sequence and cut the phosphate backbone. When two pieces of DNA are cut by the same restriction enzyme, the two ends have compatible overhangs that can be reconnected by ligase.