Preparation with amplification

Examples of Sensors Prepared with Amplification Technology... 

The variety of fiber opUc sensors prepared with amplification technology demonstrate the generality of the method. 

Fig. 30. Detection of mRNA on a membrane or in situ with labeled gene probes. A Detection of mRNA with a fluorescein-labeled single stranded nucleic acid probe, using POD-conjugated anti-fluorescein antibody. B Use of two gene probes labeled with different molecules (fluorescein and digoxigenin) and detected with specific antibodies, both coupled to AP and using two substrates, leading to differently colored products. This in situ hybridization scheme allows the simultaneous detection of two mRNA species in a tissue or cell preparation. C Amplification systems involving more than one antibody can be used to increase specificity and signal intensity.

In applications where PCR is used for the routine detection of very small quantities of nudeic adds, there is a significant problem with potential contamination. To reduce this to manageable levels, it is essential that appropriate precautions are taken. This indudes a rigorous separation of laboratory areas for sample preparation, PCR amplification and product analysis use of dedicated pipettes and pre-pre-... 

PCR-generated probes are simple to prepare. During amplification, the PGR product typically is labeled with nucleotides that are radioactive, are fluorescent, or have attached affinity labels. If desired, single-stranded probes can be obtained by using a biotin-labeled pruner, followed by soHd-phase separation with streptavidin. 

Typing methods involve the design of primer pairs that are able to amplify all alleles at the target HLA locus with the polymorphic sequence motifs situated between the primer sites. Laboratories usually amplify at least exons 2 and 3 of class I genes and exon 2 of class II genes. Some prepare larger amplification products that include exon 1 and/or exon 4 of class I genes. Amplification primers may be located within exons or introns. Primers positioned in introns allow complete analysis of exons and inspection of exon/intron junctions for splice site polymorphisms. Primers must be carefully chosen to attain locus-specific ampfification since... 

The systems described thus far addressed only a portion of the individual tasks required to elucidate DNA sequence. A more efficient process would fully integrate the tasks of DNA extraction, purification, template preparation, and amplification with sequencing reaction chemistry, post reaction cleanup, separation, and detection [152]. Several groups have explored partially integrated systems intended to streamline various steps in this process [152-156],... 

The first DNA preparations in this part of the study was PCR product - DNA of Chlamydia trachomatis 17 > bp), in the presence of a smaller by molecular mass internal control of human DNA. After migration the gel was exposured for 5, 30, 300 and 600 seconds by transilluminator Vilber Lourmat, equipped with 6 UV lamps with irradiance W = 0,24 W/m2 and 254 nm filter. The degree of structural integrity loss of amplificated DNA was evaluated by the decrease of brightness intensity of the of the bands processed by using the tools of "ImageJ" computer program. 

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