Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.

/pdf/a-new-mathematical-model-for-relative-quantification-in-real-2rwc9jj6m2.pdf

A new mathematical model for relative quantification in real-time RT-PCR.

A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

/pdf/primer-directed-enzymatic-amplification-of-dna-with-a-4w71pk8nlz.pdf

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

https://zenodo.org/record/1230922/files/article.pdf

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

Measurement of Chloramphenicol Acetyltransferase in Extracts of Mammalian Cells Using Thin-layer Chromatography.

Purification of Bacteriophage λ Particles by Pelleting/Centrifugation.

Purification of DNA Recovered from Agarose and Polyacrylamide Gels by Anion-exchange Chromatography.

The following is an elegant and simple protocol for generating and cloning blunt-ended DNA. Incubation of a ligation reaction in the presence of an excess amount of restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are regenerated when plasmid molecules ligate to themselves. The method requires that ligation of the plasmid to a target DNA molecule destroys the restriction site, so preventing the restriction enzyme from digesting recombinants generated during the ligation reaction. The net effect of constant reclamation of unit-length linear vector molecules is to drive the equilibrium of the ligation reaction strongly in favor of recombinants between vector and insert. The method is efficient because regeneration of vector DNA, ligation, and polishing the termini of PCR-generated fragments of DNA all occur simultaneously in the same reaction mixture.

Cloning Polymerase Chain Reaction (PCR) Products: Blunt-End Cloning.

In this protocol a randomly labeled single-stranded RNA probe is prepared and then hybridized to a population of mRNA molecules. The RNAs are digested with a mixture of RNase A and RNase T1. The hybrid molecules, which are resistant to the RNases, are separated and analyzed using gel electrophoresis and radiography.

Joseph Sambrook

Papers

Measurement of Chloramphenicol Acetyltransferase in Extracts of Mammalian Cells Using Thin-layer Chromatography.

Purification of Bacteriophage λ Particles by Pelleting/Centrifugation.

Purification of DNA Recovered from Agarose and Polyacrylamide Gels by Anion-exchange Chromatography.

Cloning Polymerase Chain Reaction (PCR) Products: Blunt-End Cloning.

Ribonuclease protection: mapping RNA with ribonuclease and radiolabeled RNA probes.