membrane placed on surface of agar plate remove to membrane
1. incubate filter with primary antibody
2. incubate filter with secondary antibody prot^nsbind ■ |inked to alkaline phosphatase
Jl i , add substrate for alkaline | phosphatase—a colored product is produced
•n specific bacterial colony identified
Figure 7.8. Selection of a cDNA clone with an antibody probe.
one of the strands of the cDNA. The first step is to use the genetic code to predict all possible DNA sequences that could code for a short stretch of amino acids within the protein. This strategy is shown in Figure 7.7. The sequence—met gln lys phe asn—can be coded for by 16 possible sequences, because of the redundancy of the genetic code. All 16 oligonucleotide sequences are synthesized. One of the 16 sequences will be complementary to the cDNA we want to select from the library. The oligonucleotides are tagged with a radioactive phosphate group (32P) at their 5' ends using the enzyme polynucleotide kinase (PNK) and the substrate [y-32P]ATP, that is, ATP whose y phosphate is the radioactive isotope 32P. PNK removes the 5'-phosphate group from each oligonucleotide, leaving a 5'-hydroxyl group. The enzyme then transfers the y (32P) phosphate group of [y -32P]ATP to the 5'-hydroxyl.
The nylon membrane to which the library DNA is attached is incubated together with the mixture of radiolabeled oligonucleotides. This process is called hybridization, a word used whenever two nucleic acid strands associate together by hydrogen bonding. In this case the oligonucleotide complementary in sequence to the clone we want to select will hydrogen bond to the single-stranded DNA on the nylon membrane. Once hybridization is complete, excess oligonucleotide is washed from the nylon membrane, which is now covered with a sheet of X-ray film and placed in a light-tight cassette. The radioactivity in the oligonucleotide will darken the silver grains on the X-ray film—a process known as autoradiography. A positive clone will show up as a black spot on the film. Superimposing the X-ray film back onto the original bacterial plate will identify the living bacterial colony that contains the desired foreign DNA clone.
Antibody Probes for cDNA Clones. This method makes use of specific antibodies to detect bacteria expressing the protein product of the DNA to be cloned. For this to work, the foreign DNA must be expressed in the bacterial cells; that is to say, its information must be copied first into mRNA and then into protein. To ensure efficient expression, the plasmid vector contains a bacterial promoter sequence that is used to control transcription of foreign DNA. Such cloning vectors are known as expression vectors. The promoter of the lac operon is commonly used in this way. The clone library is plated onto agar plates containing an inducer of the lac operon such as IPTG (page 114) to ensure that lots of mRNA and in turn lots of protein is synthesized. Figure 7.8 shows how an antibody, linked to an enzyme (usually alkaline phosphatase), can detect a positive clone by generating a colored product. The pattern of colored spots on the nylon membrane is used to identify the bacterial clones of interest on the original agar plate.
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