Cloning of cDNA and Genomic DNA Allows the Isolation of Defined Sequences

Messenger RNA (mRNA) isolated from cells can be transcribed into complementary DNA (cDNA) with the enzyme reverse transcriptase. The cDNA can be cloned by inserting it into a plasmid vector carrying a gene that confers resistance to an antibiotic, such as ampicillin. The resulting recombinant plasmid DNA is subsequently transferred into specially treated E. coli cells by one of several techniques; the transfer process is called transfection. If the foreign DNA is incorporated into the host cell and expressed, the cell is said to be transformed. When the cells are cultured on agar plates containing ampicillin, only transformed cells containing the ampicillin-resistance gene will survive and grow (Figure 23-7). A collection of DNA sequences within plasmid vectors representing all the mRNA sequences derived from a cell or tissue is called a cDNA library. A cDNA library differs from a genomic library (see Figure 23-8) by virtue of the fact that it contains only the sequences derived from mRNA, the sequences that represent expressed genes.

Genomic cloning, cloning of the entire genome of an animal, requires specialized vectors. E. coli plasmid vectors are impractical for cloning of all the genomic DNA fragments that constitute a large genome because of the low efficiency of E. coli transformation and the small number of transformed colonies that can be detected on a typical petri dish. Instead, cloning vectors derived from bacteriophage X are used to clone genomic DNA fragments obtained by cleaving chromosomal DNA with restriction enzymes (Figure 23-8). Bacterio-phage X DNA is 48.5 kb long and contains a central section of about 15 kb that is not necessary for X replication in E. coli and can therefore be replaced with foreign genomic DNA. As long as the recombinant DNA does not exceed the length of the original X-phage DNA by more than 5%, it can be packaged into the X-phage head and propagated in E. coli. This means that somewhat more than 1.5 X 104 base pairs can be cloned in one particle of X phage. A collection of X clones that includes all the DNA sequences of a given species is called a genomic library. lt has been calculated that about 1 million different recombinant X-phage particles would be needed to form a genomic DNA library representing an entire mammalian genome, which contains about 3 X 109 base pairs.

Often the 20-25 kb stretch of DNA that can be cloned in bacteriophage X is not long enough to include the regulatory

Ampicillin-resistance gene +

Ampicillin-resistance gene +

DNA fragment to be cloned

Plasmid vector

Enzymatically insert DNA into plasmid vector

Recombinant plasmid

Bacterial chromosome

Recombinant plasmid

Mix E. coli cells with plasmids in presence of CaCl2

Culture on nutrient agar plates containing ampicillin

Transformed Cells that do not take up

E. coli cell survives plasmid die on ampicillin plates

Independent plasmid replication

Ampicillin Plates

Colony of cells each containing copies of the same recombinant plasmid

FIGURE 23-7

cDNA cloning using a plasmid vector. A plasmid containing a replication origin and an ampicillin-resistance gene is cut with a restriction endonuclease that produces blunt ends. After addition of a poly-C tail to the 3' ends of the cDNA and of a complementary poly-G tail to the 3' ends of the cut plasmid, the two DNAs are mixed, annealed, andjoined by DNA ligase, forming the recombinant plasmid. Uptake of the recombinant plasmid into E. coli cells is stimulated by high concentrations of CaCl2. Transformation occurs with a low frequency, but the transformed cells can be selected in the presence of ampicillin. [Adapted from H. Lodish et al, 1995, Molecular Cell Biology, 3rd ed. Scientific American Books.]

Bacteriophage

Replaceable region

Partially digest with Sau3A

20 kb fragment with sticky ends

Cut with BamHI

X vector arms with sticky ends

Anneal and ligate

Recombinant X DNA

Package into X head

FIGURE 23-8

Genomic DNA cloning using bacteriophage \ as the vector. Genomic DNA is partly digested with Sau3A, producing fragments with sticky ends. The central 15-kb region of the \-phage DNA is cut out with SamHI and discarded. These two restriction enzymes produce complementary sticky ends, so the genomic and DNA fragments can be annealed and ligated. After the resulting recombinant DNA is packaged into a \-phage head, it can be propagated in E. coli.

TABLE 23-6

Vectors and maximum length of DNA that they can carry

BACs are much easier to propagate and are the vector of choice for many large-scale cloning efforts.

Maximum length of

Vector type cloned DNA (kb)

Plasmid 20

Bacteriophage \ 25

Cosmid 45

Bacteriophage P1 100

Bacterial artificial chromosome (BAC) 100-300

Yeast artificial chromosome (YAC) >1000

sequences that lie outside the 5' and 3' ends of the direct coding sequences of a gene. As noted already, larger genomic DNA fragments—between 30 and 50 kb in length—can be cloned in a cosmid vector. A recombinant cosmid vector, although not a fully functional bacteriophage, can infect E. coli and replicate as a plasmid, generating a cosmid library. Recently, a larger E. coli virus, called bacteriophage P1, has been used to package DNA fragments up to 100 kb long. Even larger DNA fragments, greater than a megabase (1000 kb) in length, can be cloned in yeast artificial chromosomes (YACs), which are linear DNA segments that can replicate in yeast cells (Table 23-6). The BAC, or bacterial artifical chromosome, is another useful vector. BACs can accept pieces of DNA up to 100-300 kb in length. Although YACs accept larger inserts of foreign DNA,

Was this article helpful?

0 0
Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

Get My Free Ebook


Responses

  • remo
    How to ligate ampicillin resistant?
    8 years ago

Post a comment