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.

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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.

Publisher Summary This chapter describes the probing structure-function relationships of tissue-type plasminogen activator by oligonucleotide-mediated site-specific mutagenesis. Tissue-type plasminogen activator (t-PA), a 66-kDa member of the chymotrypsin family of serine proteases, catalyzes the rate-limiting step in the endogenous fibrinolytic cascade: conversion of the zymogen plasminogen into the active enzyme plasmin. Plasmin, a serine protease of relatively broad specificity, efficiently degrades the fibrin that forms the meshwork of a thrombus or blood clot. The fibrinolytic cascade, therefore, provides an important counterbalance in vivo to the blood coagulation cascade. Consistent with its important regulatory role, the activity of t-PA is tightly regulated in vivo . Unlike other plasminogen activators, such as urokinase or streptokinase, t-PA both binds to and is dramatically stimulated by fibrin. The activity of circulating t-PA is also held in check by specific protease inhibitors present in human plasma. The most important endogenous inhibitor of t-PA is endothelial cell plasminogen activator inhibitor type 1 (PAI-l), a 50-kDa member of the serpin, or serine protease inhibitor, gene superfamily.

Probing structure-function relationships of tissue-type plasminogen activator by oligonucleotide-mediated site-specific mutagenesis.

This is a general protocol for the isolation of mRNA from total RNA using oligo(dT) coupled to magnetic beads. First, total RNA is dissolved in a high-salt buffer and heated briefly to 65°C-70°C, followed by immediate cooling on ice to disrupt secondary structures. The RNA is subsequently annealed to the oligo(dT)-magnetic beads at room temperature; the high-salt binding buffer stabilizes the poly(A)-oligo(dT) complexes. A high-salt washing buffer is then used to wash away unbound RNAs while retaining oligo(dT)-bound poly(A)+ mRNAs. To elute the poly(A)+ mRNAs from the beads, a low-salt buffer (or water) is used to destabilize the poly(A)-oligo(dT) complexes. Alternatively, poly(A)+ mRNAs can be retained on the beads for downstream applications (e.g., solid-phase cDNA synthesis).

Isolation of Poly(A)+ Messenger RNA Using Magnetic Oligo(dT) Beads.

Dolichol-linked oligosaccharides consisting of twoN-acetylglucosamine, nine mannose, and three glucose residues (Glc3Man9GlcNAc2) are transferred to proteins that contain the consensus sequence Asn-X-Ser/Thr. This transfer occurs upon protein import into the lumen of the endoplasmic reticulum. An intermediate in the biosynthesis of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide contains two GlcNAc and five mannose residues. This intermediate serves as a substrate for further mannosylation and glucosylation before transfer to protein. The addition of the sixth mannose residue to this intermediate requires the enzyme mannosyltransferase VI and the mannose donor, mannose-P-dolichol. Several different CHO cell line mutants that fail to efficiently catalyze this transfer have been described. In this report, we examine seven independent mutant cell lines with various biochemical phenotypes and demonstrate that all can be assigned to one of three genetic complementation groups. One mutation affects mannose-P-dolichol biosynthesis (Lec15), three affect dolichol phosphate biosynthesis (Lec9), and three appear to affect the functional orientation of enzyme substrates (PIR).

Joseph Sambrook

Papers

Probing structure-function relationships of tissue-type plasminogen activator by oligonucleotide-mediated site-specific mutagenesis.

Isolation of Poly(A)+ Messenger RNA Using Magnetic Oligo(dT) Beads.

Complementing mutant alleles define three loci involved in mannosylation of Man5-GlcNAc2-P-P-dolichol in Chinese hamster ovary cells

Rapid Amplification of 5' cDNA Ends (5'-RACE).

Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde.