Probes Search Libraries for Specific Genes or cDNA Molecules

A variety of molecules can be used to "probe" libraries in search of a specific gene or cDNA molecule or to define and quantitate DNA or RNA separated by electrophore-sis through various gels. Probes are generally pieces of DNA or RNA labeled with a 32P-containing nucleotide—or fluorescently labeled nucleotides (more commonly now). Importantly, neither modification (32P or fluorescent-label) affects the hybridization properties of the resulting labeled nucleic acid probes. The probe must recognize a complementary sequence to be effective. A cDNA synthesized from a specific mRNA can be used to screen either a cDNA library for a longer cDNA or a genomic library for a complementary sequence in the coding region of a gene. A popular technique for finding specific genes entails taking a short amino acid sequence and, employing the codon usage for that species (see Chapter 38), making an oligonucleotide probe that will detect the corresponding DNA fragment in a genomic library. If the sequences match exactly, probes 15-20 nucleotides long will hybridize. cDNA probes are used to detect DNA fragments on Southern blot transfers and to detect and quantitate RNA on Northern blot transfers. Specific antibodies can also be used as probes provided that the vector used synthesizes protein molecules that are recognized by them.

Ampicillin Tetracycline

Figure 40-4. A method of screening recombinants for inserted DNA fragments. Using the plasmid pBR322, a piece of DNA is inserted into the unique PstI site. This insertion disrupts the gene coding for a protein that provides ampicillin resistance to the host bacterium. Hence, the chimeric plasmid will no longer survive when plated on a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin can therefore be used to distinguish clones of plasmid that contain an insert. A similar scheme relying upon production of an in-frame fusion of a newly inserted DNA producing a peptide fragment capable of complementing an inactive, deleted form of the enzyme p-galactosidase allows for blue-white colony formation on agar plates containing a dye hydrolyzable by p-galactoside. p-Galactosidase-positive colonies are blue.

Figure 40-4. A method of screening recombinants for inserted DNA fragments. Using the plasmid pBR322, a piece of DNA is inserted into the unique PstI site. This insertion disrupts the gene coding for a protein that provides ampicillin resistance to the host bacterium. Hence, the chimeric plasmid will no longer survive when plated on a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin can therefore be used to distinguish clones of plasmid that contain an insert. A similar scheme relying upon production of an in-frame fusion of a newly inserted DNA producing a peptide fragment capable of complementing an inactive, deleted form of the enzyme p-galactosidase allows for blue-white colony formation on agar plates containing a dye hydrolyzable by p-galactoside. p-Galactosidase-positive colonies are blue.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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