Cloning Amplifies DNA

A clone is a large population of identical molecules, bacteria, or cells that arise from a common ancestor. Molecular cloning allows for the production of a large number of identical DNA molecules, which can then be charac terized or used for other purposes. This technique is based on the fact that chimeric or hybrid DNA molecules can be constructed in cloning vectors—typically bacterial plasmids, phages, or cosmids—which then continue to replicate in a host cell under their own control systems. In this way, the chimeric DNA is amplified. The general procedure is illustrated in Figure 40-3.

Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to the host cell. Plasmids have several properties that make them extremely useful as cloning vectors. They exist as single or multiple copies within the bacterium and replicate independently from the bacterial DNA. The complete DNA sequence of many plasmids is known; hence, the precise location of restriction enzyme

Table 40-3. Some of the enzymes used in recombinant DNA research.1

Enzyme

Reaction

Primary Use

Alkaline phosphatase

Dephosphorylates 5' ends of RNA and DNA.

Removal of 5'-PO4 groups prior to kinase labeling to prevent self-ligation.

BAL 31 nuclease

Degrades both the 3' and 5' ends of DNA.

Progressive shortening of DNA molecules.

DNA ligase

Catalyzes bonds between DNA molecules.

Joining of DNA molecules.

DNA polymerase I

Synthesizes double-stranded DNA from single-stranded DNA.

Synthesis of double-stranded cDNA; nick translation; generation of blunt ends from sticky ends.

DNase I

Under appropriate conditions, produces single-stranded nicks in DNA.

Nick translation; mapping of hypersensitive sites; mapping protein-DNA interactions.

Exonuclease III

Removes nucleotides from 3' ends of DNA.

DNA sequencing; mapping of DNA-protein interactions.

X exonuclease

Removes nucleotides from 5' ends of DNA.

DNA sequencing.

Polynucleotide kinase

Transfers terminal phosphate (y position) from ATP to 5'-OH groups of DNA or RNA.

32P labeling of DNA or RNA.

Reverse transcriptase

Synthesizes DNA from RNA template.

Synthesis of cDNA from mRNA; RNA (5' end) mapping studies.

S1 nuclease

Degrades single-stranded DNA.

Removal of "hairpin" in synthesis of cDNA; RNA mapping studies (both 5' and 3' ends).

Terminal transferase

Adds nucleotides to the 3' ends of DNA.

Homopolymer tailing.

'Adapted and reproduced, with permission, from Emery AEH: Page 41 in: An Introduction to Recombinant DNA. Wiley, 1984.

'Adapted and reproduced, with permission, from Emery AEH: Page 41 in: An Introduction to Recombinant DNA. Wiley, 1984.

Circular plasmid DNA

Linear plasmid DNA with sticky ends

Human DNA

Circular plasmid DNA

Linear plasmid DNA with sticky ends

AATT

TTAA

Piece of human DNA cut with same restriction nuclease and containing same sticky ends

Plasmid DNA molecule with human DNA insert (recombinant DNA molecule)

Figure 40-3. Use of restriction nucleases to make new recombinant or chimeric DNA molecules. When inserted back into a bacterial cell (by the process called transformation), typically only a single plasmid is taken up by a single cell, and the plasmid DNA replicates not only itself but also the physically linked new DNA insert. Since recombining the sticky ends, as indicated, regenerates the same DNA sequence recognized by the original restriction enzyme, the cloned DNA insert can be cleanly cut back out of the recombinant plasmid circle with this en-donuclease. If a mixture of all of the DNA pieces created by treatment of total human DNA with a single restriction nuclease is used as the source of human DNA, a million or so different types of recombinant DNA molecules can be obtained, each pure in its own bacterial clone. (Modified and reproduced, with permission, from Cohen SN: The manipulation of genes. Sci Am [July] 1975;233:34.)

Human DNA

AATT

TTAA

Piece of human DNA cut with same restriction nuclease and containing same sticky ends

Plasmid DNA molecule with human DNA insert (recombinant DNA molecule)

Figure 40-3. Use of restriction nucleases to make new recombinant or chimeric DNA molecules. When inserted back into a bacterial cell (by the process called transformation), typically only a single plasmid is taken up by a single cell, and the plasmid DNA replicates not only itself but also the physically linked new DNA insert. Since recombining the sticky ends, as indicated, regenerates the same DNA sequence recognized by the original restriction enzyme, the cloned DNA insert can be cleanly cut back out of the recombinant plasmid circle with this en-donuclease. If a mixture of all of the DNA pieces created by treatment of total human DNA with a single restriction nuclease is used as the source of human DNA, a million or so different types of recombinant DNA molecules can be obtained, each pure in its own bacterial clone. (Modified and reproduced, with permission, from Cohen SN: The manipulation of genes. Sci Am [July] 1975;233:34.)

cleavage sites for inserting the foreign DNA is available. Plasmids are smaller than the host chromosome and are therefore easily separated from the latter, and the desired plasmid-inserted DNA is readily removed by cutting the plasmid with the enzyme specific for the restriction site into which the original piece of DNA was inserted.

Phages usually have linear DNA molecules into which foreign DNA can be inserted at several restriction enzyme sites. The chimeric DNA is collected after the phage proceeds through its lytic cycle and produces mature, infective phage particles. A major advantage of phage vectors is that while plasmids accept DNA pieces about 6-10 kb long, phages can accept DNA fragments 10-20 kb long, a limitation imposed by the amount of DNA that can be packed into the phage head.

Larger fragments of DNA can be cloned in cosmids, which combine the best features of plasmids and phages. Cosmids are plasmids that contain the DNA sequences, so-called cos sites, required for packaging lambda DNA into the phage particle. These vectors grow in the plasmid form in bacteria, but since much of the unnecessary lambda DNA has been removed, more chimeric DNA can be packaged into the particle head. It is not unusual for cosmids to carry inserts of chimeric DNA that are 35-50 kb long. Even larger pieces of DNA can be incorporated into bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or E. coli bacteriophage Pl-based (PAC) vectors. These vectors will accept and propagate DNA inserts of several hundred kilobases or more and have largely re-

Table 40-4. Cloning capacities of common cloning vectors.

Vector

DNA Insert Size

Plasmid pBR322

0.01-10 kb

Lambda charon 4A

10-20 kb

Cosmids

35-50 kb

BAC, P1

50-250 kb

YAC

500-3000 kb

placed the plasmid, phage, and cosmid vectors for some cloning and gene mapping applications. A comparison of these vectors is shown in Table 40-4.

Because insertion of DNA into a functional region of the vector will interfere with the action of this region, care must be taken not to interrupt an essential function of the vector. This concept can be exploited, however, to provide a selection technique. For example, the common plasmid vector pBR322 has both tetracycline (tet) and ampicillin (amp) resistance genes. A single PstI restriction enzyme site within the amp resistance gene is commonly used as the insertion site for a piece of foreign DNA. In addition to having sticky ends (Table 40-2 and Figure 40-2), the DNA inserted at this site disrupts the amp resistance gene and makes the bacterium carrying this plasmid amp-sensitive (Figure 40-4). Thus, the parental plasmid, which provides resistance to both antibiotics, can be readily separated from the chimeric plasmid, which is resistant only to tetracycline. YACs contain replication and segregation functions that work in both bacteria and yeast cells and therefore can be propagated in either organism.

In addition to the vectors described in Table 40-4 that are designed primarily for propagation in bacterial cells, vectors for mammalian cell propagation and insert gene (cDNA)/protein expression have also been developed. These vectors are all based upon various eukary-otic viruses that are composed of RNA or DNA genomes. Notable examples of such viral vectors are those utilizing adenoviral (DNA-based) and retroviral (RNA-based) genomes. Though somewhat limited in the size of DNA sequences that can be inserted, such mammalian viral cloning vectors make up for this shortcoming because they will efficiently infect a wide range of different cell types. For this reason, various mammalian viral vectors are being investigated for use in gene therapy experiments.

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