Polymerase chain reaction and other methods to detect hot-spot and multiple gene mutations.

Gene mutations responsible for main genetic diseases as Duchenne/Becker muscular dystrophy or cystic fibrosis, and involved in more important diseases like cancer or cardiac diseases have been identified. Direct DNA tests can now be performed for these disorders. However, despite the knowledge of the exact alteration of the DNA sequence in these diseases, incorporating DNA analysis into large screening programs has been hindered by technical difficulties since each different mutation requires a different probe to be detected. To overcome these problems the polymerase chain reaction technique (PCR) has been proposed. Multiplex PCR procedure is possible and consists of simultaneously amplifying several separate DNA sequences (the upper limit of the number of multiplex reactions that can be executed at one time is not known; eight or nine separate DNA sequences amplified have already been described). Another approach is to directly sequence the gene mutations generally concentrated in 'hot-spot' regions. This way it is possible to identify numerous mutations using only one micro-sequencing reaction (50-100 nucleotides). New generations of very sophisticated systems like capillary electrophoresis or non-isotopic, but very sensitive, microsequencing systems should allow in a near future to withdraw the use of PCR for this purpose.

Non-isotopic probe labelling: advantages and drawbacks for diagnostic specifications.

Nucleic acid probes represent an essential tool for molecular investigation in both basic and applied research. In this last field they are particularly useful for diagnosis or prognosis and even for therapy. In the diagnosis, the nucleic probes theoretically allow to reach a sensitive level of reliability never obtained before. Nevertheless, the use of nucleic probes in diagnosis routine need to respect a drastic schedule of conditions since: nucleic probes have to be perfectly specific. nucleic probes have to be sensitive and to allow the detection of less than 100 femtogram of nucleic acid target in routine. In some cases this threshold has to be less than 1 femtogram (for instance HIV detection cases). Nucleic probes have to be absolutely reliable without risk to obtain false positives or negatives. Complete hybridization results must be obtained fast (within 2 or 3 hours in most cases). Labelling systems of nucleic acids must be non-isotopic. Numerous labelling systems, isotopic or non isotopic, have been described during the last fifteenth years. Since some time, the non-isotopic labelling is allowing to contemplate a widespread diagnostic use of it in daily medical, veterinary and agronomy practice. At the present time, some old labelling systems are ahead and new systems are going to be developed worldwide. The future of nucleic probes for the diagnosis needs to assume the schedule of conditions described above. In addition, uses and results obtained with these probes have to be standardized (what is not done yet). Automation and use of amplification of target nucleic acids must be viewed for some applications.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of phosphoenolpyruvate and ribulose 1,5-bisphosphate carboxylase transcripts in maize leaves by in situ hybridization with sulfonated cDNA probes.

This report outlines an efficient in situ hybridization method for locating specific mRNAs in tissue cryosections using sulfonated cDNA probes. The method involves chemical modification of DNA probes by insertion of a sulfone radical on cytosine residues, which generates a specific epitope. Sulfonated DNA is then detected by using indirect immunochemical procedure. Alternatively, antibodies conjugated to fluorescein or to alkaline phosphatase were used for mRNA detection. In situ hybridization was developed to study aspects of mesophyll and bundle sheath cell differentiation in maize leaves. Our results indicate that phosphoenolpyruvate carboxylase (PEP C) mRNA is restricted to mesophyll cells, and the nucleus-encoded mRNA of the small subunit (SSU) ribulose 1,5-bisphosphate carboxylase (RuBP C) is limited to the cytosol of bundle sheath cells. Thus, using in situ hybridization, we have demonstrated that the differential distribution of PEP C and RuBP C proteins in the two cell types also reflects the location of their mRNAs. These data imply either a tissue-specific transcriptional regulation or a selective mRNA degradation.

A new sensitive non-isotopic method using sulfonated probes to detect picogram quantities of specific DNA sequences on blot hybridization.

The use of non-radioactive systems to detect target DNA or RNA displays many advantages such as safe manipulation, potential use in non-specialized scientific area and prolonged lifetime of the probes (one year or more). We here describe a method we have improved and optimized using sulfonated DNA probes for hybridization on dot and Southern blots. Sulfonation is an easy chemical modification procedure which does not require enzymatic coctail as does nick-translation. Sensitivity of this method has been particularly improved by using a new blocking solution, containing heparin, which allows easy and fast detection of picogram quantities of DNA. This method allows the use of nitrocellulose as well as nylon membranes with very low background. Equal resolution is obtained in comparative experiments involving both sulfonated and 32P-radiolabelled probes. Single copy gene sequences are readily detected in nuclear DNA. These results allow the use of this procedure for restriction fragment length polymorphism (RFLP) studies.

Comparative macromolecular analysis of the cytoplasms of normal and cytoplasmic male sterile Brassica napus.

Chloroplast (cp) and mitochondrial (mt) compartments of normal (N) and cytoplasmic male sterile (cms) lines of Brassica napus have been characterized and compared on the basis of cp and mt DNA restriction enzyme analysis and in vitro protein synthesis by isolated mitochondria. Cytoplasmic male sterility of B. napus (rape) comes from cms Raphanus sativus (radish) through intergeneric crosses.Cp DNAs isolated from N and cms lines had distinct restriction patterns with Sal I, Kpn I and Sma I enzymes. The size of the two cp DNAs measured from the restriction patterns was found to be identical and of about 95 × 10(6) d. N and cms lines of B. napus were characterized by specific mt DNAs, as shown from Sal I, Kpn I, Pst I and Xho I cleavage patterns. The small number of well-separated restriction fragments obtained with Sal I enabled us to determine precisely mt DNA sizes. The values of 136.5 and 140.3 × 10(6) d, obtained from restriction patterns with N and cms DNAs respectively, are smaller than any of those previously obtained from studies on other genera. With molecular hybridization experiments, it was possible to distinguish N and cms lines by the different locations of rRNA genes on the cp and mt DNAs.Two lines of B. napus are characterized by specific mt translation products formed in isolated mitochondria.

Cytoplasmic DNA variation and relationships in cereal genomes.

Chloroplast (cp) and mitochondrial (mt) DNAs were isolated from four cereal genomes (cultivated wheat, rye, barley and oats) and compared by restriction nuclease analysis. Cleavage of cp and mt DNAs by Sal I, Kpn I, Xho I and EcoR I enzymes indicated that each cereal group contains specific cytoplasmic DNAs. A phylogenetic tree of cereal evolution has been obtained on the basis of cp DNA homologies. It is suggested that wheat and rye diverged after their common ancestor had diverged from the ancestor of barley. This was preceded by the divergence of the common ancestor of wheat, rye and barley and the ancestor of oats.The molecular weight of the different cp DNAs was determined from the Sal I and Kpn I patterns. cp DNAs from wheat, rye, barley and oats appeared to be characterized by a very similar molecular weight of about 80-82.10(6) d.In the case of the mt DNAs, the great number of restriction fragments obtained with the restriction enzymes used prevented precise comparisons and determination of molecular weights.