Genetic analysis, techniques and applications最新文献

We describe an enrichment of foetal cells from maternal blood with a combination of double density gradient and Magnetic Activated Cell Sorting (MACS) of CD71, glycophorin A (GPA), CD34 and CD36 antibodies labeled cells followed by fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes for determination of foetal sex.

Extraction of nuclei from unmounted archived tissue has become a method widely used for molecular cytogenetic investigations. It is a suitable approach to take advantage of the large series of formalin fixed and subsequently paraffin-embedded material which has been collected in the times before interphase cytogenetic analysis has become possible. We present a new kind of assay for the extraction of nuclei from one single mounted tissue section; the extracted interphase nuclei are suitable very well for a fluorescence in situ hybridization (FISH) approach. The method described has been successfully used for the analysis of the INT2/FGF3-amplicon in 20 samples of oral squamous cell carcinoma.

A new method of amplifying short DNA molecules immobilized on a solid support has been developed. This method uses a solid-phase rolling circle replication reaction, termed rolling circle amplification (RCA). The probe consists of a single-stranded DNA primer anchored at the 5′ terminus to a solid support and a single stranded DNA template hybridized to the immobilized primer. Here, DNA ligase was used to circularize the template, and DNA polymerase I was used to extend the immobilized primer in a rolling circle replication reaction. This method was used to identify a known polymorphism in BRCA1 exon 5. These results demonstrate that RCA offers considerable promise to facilitate effective mutation screening of DNA using a solid-phase format.

A simple and rapid PCR-based method for screening transformed Arabidopsis plants has been developed. Based on the quantity of chlorophyll present in a protoplast suspension, the optimal amount of template is calculated and a fragment of the transgene is amplified.

Taking advantage of highly conserved domains present in the secA gene from Escherichia coli and Bacillus subtilis, we designed degenerate oligonucleotides (oligos) corresponding to these regions. These oligos were used as primers in PCR in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA. The PCR product was used as a probe to recover genomic fragments from a λ library of Br. flavum MJ233. The complete nucleotide sequence (nt) of the cloned 5.3-kb EcoRI fragment containing the secA homolog from Br. flavum MJ233 indicated that the deduced gene product of the Br. flavum secA homolog is composed of 845 amino acids (aa) with a deduced molecular weight (MW) of 95 429. Comparison of this aa sequence to the corresponding sequences from E. coli and B. subtilis revealed a high degree of conservation and suggested that the Br. flavum secA homolog has putative ATP binding regions.

Nucleotide sequence analysis of arbitrarily primed PCR products from two known regions of human genome revealed that at least six contiguous bases at the 3′-end of a primer of 20 bases were perfectly matched in the primer-template hybrids.

Total RNA was extracted from venom glands of Agkistrodon acutus. The cDNA encoding Lys-49 phospholipase A₂ (PLA₂) was amplified by reverse transcriptional polymerase chain reaction (RT-PCR). The cDNA was cloned into the pGEMT-vector and sequenced. The open reading frame (ORF) of Lys-49 PLA₂ consists of 414 bp encoding 138 amino acids, which includes a signal peptide of 16 amino acids and a matured peptide of 122 amino acids. It shows 76% identity in amino acids with another reported Lys-49 PLA₂. Because residue 49 in mature peptide is Lysine, it probably possesses myotoxicity. These results indicate there are at least two kinds of myotoxin in the venom of A. acutus.

The properties of human DNA fingerprints detected by multilocus polymeric monocore probes (PMC probes) have been investigated. The PMC probes were produced by the polymerase chain reaction with two partly complementary oligonucleotides homologous to various minisatellite or microsatellite core sequences (Ijdo J, Wells RA, Baldini A, Reeders ST. Nucleic Acids Res 1991;19:4780). It has been shown that these probes possess increased sensitivity, they detect considerably more hypervariable fragments in genomic DNA thus exhibiting advantages over the corresponding oligonucleotides and natural polycore minisatellite probes. Variation in the DNA fingerprints of different individuals produced by these probes indicates that the probability of accidental identity is very low (<10⁻¹²). According to the data of cross-hybridization with PMC probes of various specificity, several distinct families can be distinguished among G-rich hypervariable sequences of the human genome.

Genotyping of the dentatorubral-pallidoluysian atrophy (DRPLA) locus in six patient samples, representing four normal individuals and two DRPLA patients, was successfully obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). DRPLA is a dominantly inherited neurodegenerative disorder associated with the expansion of an unstable trinucleotide (CAG) repeat. The accurate determination of repeat length utilizing MALDI supports the use of this methodology for the analysis of genes containing unstable CAG trinucleotide repeats.

Large-scale screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5′ nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5′ nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes.