H A A T Tc

▲ FIGURE 9-11 Ligation of restriction fragments with complementary sticky ends. In this example, vector DNA cut with EcoRI is mixed with a sample containing restriction fragments produced by cleaving genomic DNA with several different restriction enzymes. The short base sequences composing the sticky ends of each fragment type are shown. The sticky end on the cut vector DNA (a') base-pairs only with the complementary sticky ends on the EcoRI fragment (a) in the genomic sample. The adjacent 3'-hydroxyl and 5'-phosphate groups (red) on the base-paired fragments then are covalently joined (ligated) by T4 DNA ligase.

blunt DNA ends. However, blunt-end ligation is inherently inefficient and requires a higher concentration of both DNA and DNA ligase than for ligation of sticky ends.

E. coli Plasmid Vectors Are Suitable for Cloning Isolated DNA Fragments

Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell's chromosomal DNA. These extrachromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g., yeast), exist in a parasitic or symbiotic relationship with their host cell. Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division. During cell division, copies of the plasmid DNA segregate to each daughter cell, assuring con-

tinued propagation of the plasmid through successive generations of the host cell.

The plasmids most commonly used in recombinant DNA technology are those that replicate in E. coli. Investigators have engineered these plasmids to optimize their use as vectors in DNA cloning. For instance, removal of unneeded portions from naturally occurring E. coli plasmids yields plasmid vectors, «1.2-3 kb in circumferential length, that contain three regions essential for DNA cloning: a replication origin; a marker that permits selection, usually a drug-resistance gene; and a region in which exogenous DNA fragments can be inserted (Figure 9-12). Host-cell enzymes replicate a plasmid beginning at the replication origin (ORI), a specific DNA sequence of 50-100 base pairs. Once DNA replication is initiated at the ORI, it continues around the circular plasmid regardless of its nucleotide sequence. Thus any DNA sequence inserted into such a plasmid is replicated along with the rest of the plasmid DNA.

Figure 9-13 outlines the general procedure for cloning a DNA fragment using E. coli plasmid vectors. When E. coli cells are mixed with recombinant vector DNA under certain conditions, a small fraction of the cells will take up the plas-mid DNA, a process known as transformation. Typically, 1 cell in about 10,000 incorporates a single plasmid DNA molecule and thus becomes transformed. After plasmid vectors are incubated with E. coli, those cells that take up the plasmid can be easily selected from the much larger number of cells. For instance, if the plasmid carries a gene that confers resistance to the antibiotic ampicillin, transformed cells

HindIII /\

San / XbaI BamHI I I SmaI \ \ KpnI \ \ Sad \ EcoRI \/

Plasmid cloning vector

Polylinker

Plasmid cloning vector

▲ FIGURE 9-12 Basic components of a plasmid cloning vector that can replicate within an E. coli cell. Plasmid vectors contain a selectable gene such as ampr, which encodes the enzyme p-lactamase and confers resistance to ampicillin. Exogenous DNA can be inserted into the bracketed region without disturbing the ability of the plasmid to replicate or express the ampr gene. Plasmid vectors also contain a replication origin (ORI) sequence where DNA replication is initiated by host-cell enzymes. Inclusion of a synthetic polylinker containing the recognition sequences for several different restriction enzymes increases the versatility of a plasmid vector. The vector is designed so that each site in the polylinker is unique on the plasmid.

+ DNA fragment to be cloned

Enzymatically insert DNA into plasmid vector

Recombinant plasmid

Recombinant plasmid

E. coli chromosome

Mix E. coli with plasmids in presence of CaCl2; heat pulse

Culture on nutrient agar plates containing ampicillin

Transformed cell survives

Transformed cell survives

Culture on nutrient agar plates containing ampicillin

Cells that do not take up plasmid die on ampicillin plates

Cells that do not take up plasmid die on ampicillin plates

Plasmid replication

Cell multiplication

Cell multiplication

Colony of cells, each containing copies of the same recombinant plasmid

▲ EXPERIMENTAL FIGURE 9-13 DNA cloning in a plasmid vector permits amplification of a DNA fragment.

A fragment of DNA to be cloned is first inserted into a plasmid vector containing an ampicillin-resistance gene (amp), such as that shown in Figure 9-12. Only the few cells transformed by incorporation of a plasmid molecule will survive on ampicillin-containing medium. In transformed cells, the plasmid DNA replicates and segregates into daughter cells, resulting in formation of an ampicillin-resistant colony.

can be selected by growing them in an ampicillin-containing medium.

DNA fragments from a few base pairs up to «20 kb commonly are inserted into plasmid vectors. If special precautions are taken to avoid manipulations that might mechanically break DNA, even longer DNA fragments can be inserted into a plasmid vector. When a recombinant plasmid with an inserted DNA fragment transforms an E. coli cell, all the antibiotic-resistant progeny cells that arise from the initial transformed cell will contain plasmids with the same inserted DNA. The inserted DNA is replicated along with the rest of the plasmid DNA and segregates to daughter cells as the colony grows. In this way, the initial fragment of DNA is replicated in the colony of cells into a large number of identical copies. Since all the cells in a colony arise from a single transformed parental cell, they constitute a clone of cells, and the initial fragment of DNA inserted into the parental plasmid is referred to as cloned DNA or a DNA clone.

The versatility of an E. coli plasmid vector is increased by incorporating into it a polylinker, a synthetically generated sequence containing one copy of several different restriction sites that are not present elsewhere in the plasmid sequence (see Figure 9-12). When such a vector is treated with a restriction enzyme that recognizes a restriction site in the polylinker, the vector is cut only once within the polylinker. Subsequently any DNA fragment of appropriate length produced with the same restriction enzyme can be inserted into the cut plasmid with DNA ligase. Plasmids containing a polylinker permit a researcher to clone DNA fragments generated with different restriction enzymes using the same plas-mid vector, which simplifies experimental procedures.

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment