Cloned probe

As it is thought that modifications of membranes are important in the cold stress response, a question which has not been addressed yet is the subcellular localisation of the cold-regulated gene products. No clear experimental data are available. However, from the cDNA sequences determined so far it can be deduced that the predicted proteins lack obvious signal sequences and are therefore very likely to be localised in the cytoplasm (Schaffer Fischer, 1988 Cattivelli Bartels, 1990 Kurkela Franck, 1990). These data are in agreement with comparisons of in vivo and in vitro synthesised protein patterns, because for many polypeptides no post-translational modifications were predicted from 2-dimensional electrophoresis analyses (Cattivelli Bartels, 1989). With the availability of cloned probes this problem can be analysed directly by the functional expression of the cDNA clones and the production of antibodies which can be used for immunolocalisation of the corresponding proteins. 

Cloned probes consist of a known segment of DNA inserted into a plasmid vector that is propagated by growth in a bacterium. Many different plasmid vectors are now available pBR322 was one of the first in common use. The probe may consist of the entire plasmid DNA (insert plus vector sequences), or the insert may be purified from the vector sequences. The latter method is obviously more cumbersome but may result in reduced background. The resulting probe is a double-stranded DNA probe and it must be denatured before use. 

SB Synthetic oligonucleotide probes SSb PCR-generated probes Cloned probes... 

Typically we use between 1 and 5 ng/pL of probe/slide for cloned probes. Increasing the probe concentration tends to result in higher levels of background rather than stronger in situ hybridization signal. 

Nothern blot and dot blot hybridization of R. rubrum RNA Nothern blots of the RNA from R. nibrum cells grown in different conditions when hybridized with the cloned probe for rubisco gave the following... 

In the human cell there are 23 pairs of chromosomes containing approximately 3000 million base pairs of DNA. Short sequences of DNA, perhaps with as few as 20 nucleotide units and sometimes radiolabeled, can be obtained either by chemical synthesis (gene machine) or from cloning. These short sequences can be used to probe for a complementary sequence by looking for the position to which they bind to any DNA sample under investigation, from blood for example. Such probes can detect as little as 100 fg of DNA and are the basis of forensic genetic fingerprinting tests. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 13.15 depicts such an expression vector. Linearized recombinant vector DNA is transcribed in vitro using SPG RNA polymerase. Large amounts of RNA product can be obtained in this manner if radioactive ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made. 

The induction of HSPs correlates with the increase of the levels of their transcripts. Liquid hybridisation studies conducted by Schoffl Key (1982) using cloned cDNA probes have revealed that about 20 different species of HSP18 mRNA accumulate to 19 000 copies per cell within two hours of heat... 

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