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Recombinant DNA
Cloning Technology
1. The goal of molecular cloning is large amounts of pure
DNA that can be further manipulated and studied.
2. Summary of the procedure:
a. Isolate DNA from the organism.
b. Use restriction enzymes to cut the DNA, and ligate fragments
into a cloning vector.
c. Transform recombinant DNA into a host, which will replicate
the DNA (molecular cloning) and pass copies to all progeny.
1. Restriction endonucleases (restriction enzymes) each recognize a
specific DNA sequence (restriction site), and break a phosphodiester
linkage between a 3’ carbon and phosphate within that sequence.
2. Restriction enzymes are used to create DNA fragments for cloning, and
to analyze positions of restriction sites in cloned or genomic DNA.
3. Restriction enzymes are a bacterial defense against viral DNA.
Restriction sites in the bacterial chromosome are methylated, and thus
protected.
4. A restriction enzyme has a threeletter name derived from the genus
and species of the organism from which it was isolated; it is underlined
or italicized. Roman numerals and sometimes letters designating a
particular bacterial strain may follow.
5. Many restriction sites are palindromes of 4, 6 or 8base pairs, but
others are not completely symmetrical (Figure 7.1 and Table 7.1).
b. A selectable marker, such as antibiotic resistance.
c. Unique restriction sites, so that a particular restriction enzyme cuts only once in the plasmid. A fragment of
insert DNA cut with the same enzyme is commonly inserted into the unique restriction site.
3. An example of a typical E. coli cloning vector is pUC19 (2,686bp). The pUC19 plasmid features:
a. High copy number in E. coli, with nearly a hundred copies per cell, provides a good yield of cloned DNA.
b. Its selectable marker is ampR.
c. It has a cluster of unique restriction sites, called the polylinker (multiple cloning site).
d. The polylinker is part of the lacZ (βgalactosidase) gene. The pUC19 plasmid will complement a lacZ E. coli,
allowing it to become lacZ+. When DNA is cloned into the polylinker, lacZ is disrupted, preventing
complementation from occurring.
e. Xgal, a chromogenic analog of lactose, turns blue whenβgalactosidase is present, and remains white in its
absence, so bluewhite screening can indicate which colonies contain recombinant plasmids (Figure 7.4).
5. Many plasmid cloning vectors are available, with features including different arrays of
unique restriction sites in the polylinker, and phage promoters (e.g., T7, T3, SP6) that
can be used to control transcription of the cloned DNA.
6. Plasmid cloning vectors are available for many prokaryotic and eukaryotic organisms. In
some cases the plasmids are unable to replicate, but are maintained because they
integrate into the genome.
7. Size of the insert DNA is limited in plasmid cloning vectors, and plasmids carrying more
than 5–10 kb are often unstable.
台大農藝系 遺傳學 601 20000 Chapter 7 slide 11
Fig. 7.5 Insertion of a piece of DNA into the plasmid cloning vector pUC19
1. Cosmids are constructed vector with features from both
plasmids and phages. These features include:
• An E. coli ori sequence.
• A selectable marker such as ampR
• Convenient restriction sites.
• A phage cos site, allowing the DNA to be pakaged into a
phage head for introduction into E. coli.
• Cosmid as small as 5kb are available, and 3247kb of
DNA can be inserted into them. Recombinant cosmids
< 37kb or > 52 kb cannot be packaged, don’t enter E.
coli, and therefore are not replicated.
1. A cloning vector capable of replicating in two or
more types of organism (e.g., E. coli and yeast) is
called a shuttle vector. Shuttle vectors may
replicate autonomously in both hosts, or integrate
into the host genome
1. BACs are used for cloning fragments up to about 200 kb
in E .coli. BAC vectors contain:
a. the ori of an E. coli plasmid called the F factor.
b. A multiple cloning sites.
c. A selectable marker.
d. Other features, “bells and whistles.”
2. BAC can be handles like regular bacterial plasmids, but
the F factor ori keeps copy number at one BAC molecule
per cell.
1. It is sometimes useful to have a genomic library, a
collection of clones containing at least one copy
of every DNA sequence in a genome. Genomic
libraries are available for many organisms.
2. Chromosome libraries are collections of cloned
fragments of individual chromosomes.
3. Complementary DNA (cDNA) libraries are
cloned collections of DNA copied from a cell’s
mRNA.
1. Screening the genomic library of an organism
with a large genome is laborious. Screening time
can be reduced if a gene has been localized to a
chromosome, by examining a library made from
only that chromosome. Human, for example,
have 24 different chromosome libraries (22
autosomes, X and Y).
2. Separating chromosome so they may be
individually cloned is accomplished with
techniques such as flow cytometry.
台大農藝系 遺傳學 601 20000 Chapter 7 slide 26
cDNA Libraries
1. A cell’s mRNA molecules can be copied to make complementary DNA
strands (cDNA) and the cDNA can then be cloned, creating a library
representing only the genes being expressed in the cells at that time.
2. The cDNA derives only from mature mRNA. Introns are not present.
3. The poly(A) tail at the 3’ end of the mRNA is useful for:
a. Isolating mRNA from cell lysates by passage over an oligo(dT)
column.
i. The mRNA’s poly(A) tail sticks to the poly(T) attached to the
column substrate.
ii. Other molecules pass through the column, but mRNAs are
retained.
iii. mRNAs are eluted with decreasing ionic strength buffer, resulting
in significant purification.
b. Priming the synthesis of cDNA, by providing a known 5’ sequence.
1. A number of techniques have been developed for
identifying the clone of interest in a cDNA
library. Some are discussed here.
Using Heterologous Probes
1. It is possible to identify specific genes in a
genomic library using cloned equivalent genes
(heterologous probes) from other organisms,
especially if the gene is highly conserved or the
species are closely related.
Animation: Dideoxy DNA Sequencing
1. Once cloned, DNA fragments may be sequenced,
allowing identification of gene and regulatory
sequences, and comparison with homologous
genes from different organisms.