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.

Purification of plasmid DNA by chromatography.

Dot and slot hybridization of purified RNA.

A recent report based on 68 families, including 17 with mutations in BRCA1, suggested that there was an excess of female offspring born to BRCA1 mutation carriers. We have examined the gender ratio among offspring of 511 mutation carriers from 116 BRCA1 families, 77 and 39 from Australia and the United States, respectively. We found no evidence for a significant deviation from the expected proportion of female offspring in the Australian pedigrees, but there was an excess of female offspring in pedigrees from the USA. Ascertainment bias probably explains this bias, rather than a link with X-chromosome inactivation as previously suggested, because the families from the USA were ascertained for the purposes of linkage studies whereas those from Australia were ascertained through Familial Cancer Clinics to which they had been referred for clinical genetic counseling and mutation testing.

Ratio of male to female births in the offspring of BRCA1 and BRCA2 carriers

In this protocol, oligonucleotides that are heterogeneous in sequence serve as primers for the initiation of DNA synthesis on single-stranded templates. Labeled dNTPs ([α-32P]dNTPs or biotin-, DIG-, or fluorescein-labeled dUTPs) are incorporated into the new DNA by the Klenow fragment of Escherichia coli DNA polymerase I (Pol I). The method can be adapted to radiolabel DNA in slices cut from gels cast with low-melting-temperature agarose.

Joseph Sambrook

Papers

Purification of plasmid DNA by chromatography.

Dot and slot hybridization of purified RNA.

Ratio of male to female births in the offspring of BRCA1 and BRCA2 carriers

Random Priming: Labeling of Purified DNA Fragments by Extension of Random Oligonucleotides.

Assay for β-galactosidase in Extracts of Mammalian Cells.