A gene library can be kept in phages

Figure 22.3 shows the insertion of cDNA into the DNA of a X phage. In the example shown here, the phage DNA possesses a cleavage site for the restriction endonuclease EcoRI (section 20.3). The cDNA double strand is first methylated by an EcoRI methylase at its own EcoRI restriction sites in order to protect this restriction site within the cDNA. DNA ligase is then used to link chemically synthesized double-stranded oligonucleotides with an inbuilt restriction site (in this case for EcoRI) to both ends of the double-stranded cDNA. These oligonucleotides are called linkers. The restriction endonuclease EcoRI cleaves this linker as well as the X phage DNA and thus generates sticky ends at which the complementary nucle-otides of the cDNA and the phage DNA can anneal by base pairing. The DNA strands are then linked by DNA ligase, and in this way the cDNA is inserted into the phage vector.

The phage DNA with the inserted cDNA is packed in vitro into a phage protein coat (Fig. 22.4), using a packing extract from phage-infected bacteria. In this way one obtains a gene library, in which the cDNA from many different mRNAs of the leaf tissue are packed in phages. After infecting bacteria they can be amplified ad libitum, whereby each packed cDNA is one individual clone harboring one plant gene.

The bacteria are infected by mixing them with the phages. At first bacteria grow on the agar plates to produce a bacterial lawn, and then the phages, which have been priorly propagated in bacteria, are added on top. After incubation, the infected and lysed bacterial colonies appear on the agar plate as clear spots within the bacterial lawn and are called plaques. These plaques contain newly formed phages, which can be multiplied further. It is customary to plate a typical cDNA gene library on about 10 to 20 agar plates of ca. 20 cm diameter. Ideally, each of these plaques contains only one clone. From cDNA

EcoRI methylase

Linker —-

•ch3

i

T4 DNA ligase ^ i

X-Phage DNA (Insertion vector)

G A A T T C

G A A T T C

G A A T T C

C T T A A G

c T T A A G

c T T A A G

EcoRI Î

t

Î

linker

EcoRI

EcoRI

|a A t t c

G

ÍG

C T T A A

Sticky

end

T4 DNA ligase

Figure 22.3 Insertion of cDNA into a X-phage insertion vector.

these plaques, the phage clone containing the cDNA of the desired gene is selected, using specific probes as will be described later.

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