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Thank you Jonathan Ferrand from Hudson Institute of Medical Research, VIC Australia, for a great review on our MyTaq™ Red Mix
"We have been using the MyTaq™ Red Mix for the past two years in our routine PCRs for mice genotyping, cloning/amplicon validation and standard curve preparation for our RTqPCR. This product is very convenient with gel loading buffer included, and in our hands work as well as competitor products with as low as 5ul of Mix per reaction (10ul final volume). The competitive pricing makes it a very attractive alternative for routine PCRs."
Click the link to learn more about >> MyTaq™ Red Mix
An alternative way for RNAi libraries to modify the mammalian genome on a large-scale may lie in the use of genome editing technologies such as the use of targeted endonucleases. Such a technique includes Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly-interspaced, short palindromic repeats/CRISPR-associated endonuclease cas9 (CRISPR/Cas9). CRISPR is a Cas9 endonuclease–based method for sequence-specific genome modification. It can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA.
These technologies have proven highly useful for gene editing genomes of various species and the speed at which CRISPR techniques have been exploited has been incredibly rapid.
In January 2013 researchers at MIT, the Broad Institute, and Rockerfeller University first reported the new technique based on the CRISPR/Cas9 system, for precisely altering the genomes of living cells through gene addition or deletion. Researchers say the technology could offer an easy-to-use and more cost-effective way to engineer organisms that produce biofuels; to design animal models to study human disease; or to develop new therapies, among other potential applications.
Bioline reagents have been used to augment the workflow in these exciting gene editing technologies to elucidate CRISPR systems, including our SensiFAST™ range of Real-Time PCR Kits, as well as our performance PCR polymerases.
In the first examples of genes that mediate the inhibition of a CRISPR/Cas system, researchers from the University of Toronto identified five distinct ‘anti-CRISPR’ genes in the genomes of bacteriophages infecting Pseudomonas aeruginosa.
Bondy-Denomy, J et al., (2012). Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429-432.
Researchers from Minnesota University reported precise, high-frequency editing of 15 genes in pig, goat, and cattle genomes using CRISPR/Cas and TALENs technology. RFLP analysis of colonies were analysed by PCR using MyTaq Red Mix.
Wenfang, T.W. et al., (2013). Efficient nonmeiotic allele introgression in livestock using custom endonucleases. PNAS 110(41), 16526-16531.
To learn more about performance PCR reagents from Bioline, just visit www.bioline.com
Autism spectrum disorders (ASDs), or pervasive developmental disorders (PDDs), are a category of complex developmental brain disorders that appear in early childhood, usually before the age of three. ASDs cause difficulties in social, communication and behavioural skills, but affected individuals can manifest a wide range of symptoms, from very mild to severe - hence the term "spectrum".
One in 110 children in the US has an ASD, with four times as many boys than girls being affected. In the last decade, diagnoses have increased tenfold.
ASDs are currently defined as five disorders:
- Autistic disorder (also called “classic autism”)
- Asperger syndrome
- Pervasive developmental disorder not otherwise specified (PDD-NOS)
- Rett disorder
- Childhood disintegrative disorder (CDD).
Copy number variants (CNVs) – a form of structural variation - are deletions or duplications of genomic segments ranging from several thousand to a few million base pairs. A number of population-based studies have demonstrated that CNVs can affect as much as 12% of the human genome. Large scale studies have gone on to show the importance of CNVs in determining human phenotypic variation and disease susceptibility.
There is strong evidence that rare CNVs play a role in susceptibility to ASDs. A large study published in Nature by the Autism Genome Project (AGP), an international consortium of scientists from more than 60 institutions in 12 countries, compared the DNA of almost 1000 children with ASDs and healthy children, using 1 million single-nucleotide polymorphism (SNP) arrays. The study showed that rare CNVs, possibly acting in tandem, play a significant role in the genetic aetiology of this condition. The rare variants were identified in less than 1% of the normal population but strikingly, occurred almost 20% more frequently in ASD children.
Further recent research has focused on how CNVs mediate a phenotypic effect by altering gene expression levels, which is the focus of the study by Luo and colleagues published in the June 2012 edition of the American Journal of Human Genetics. A second paper from Holt and colleagues published in the May 2012 edition of the European Journal of Human Genetics investigates an alternative mechanism whereby CNVs combine the 5' and 3' ends of two genes, creating a novel 'fusion gene'.
This pair of papers sheds light on some of the genomic imbalances that could contribute to ASD pathogenesis. Bioline offers products ideal for use in genome-wide studies of structural variants and disease susceptibility.
Luo and colleagues used genome-wide transcriptome profiling to evaluate the functional consequences of rare structural CNVs in ASD. The UCLA team along with collaborators from Yale, Wellcome Trust Sanger Institute, Carnegie Mellon, and University of Pittsburgh identified a number of interesting candidate ASD loci at 12p11.22, 15q23, 1p34.3, 3q27, and 3p26.2. For example, the 3p26.2 loci, found deleted, harbours three genes: ITPR1, SETMAR and SUMF1, all of which are down-regulated. Although none of these genes has been previously associated with autism, they are all functionally linked to the nervous system.
This study provides evidence that pathogenic structural variants have a functional impact via transcriptome alterations in ASDs at a genome-wide level. The authors also demonstrate the usefulness of integrating gene expression and mutation data to prioritize candidate genes disrupted by potentially deleterious alterations.
Luo R., et al. Am. J. Hum. Gen. 91(1): 38–55 (2012) – Genome-wide Transcriptome Profiling Reveals the Functional Impact of Rare De Novo and Recurrent CNVs in Autism Spectrum Disorders
Fusion-proteins play an important role in cancer genetics, as exemplified by BCR-ABL in leukemia and TMPRSS2-ERG in prostate cancers. A team from the Wellcome Trust Centre for Human Genetics, University of Oxford addressed the question of how common CNV-induced fusion transcripts are and whether they may play a role in ASD susceptibility. Firstly, using the published Illumina 1 million SNP array data from the AGP consortium, they performed bioinformatic analysis of existing CNV calls. They then validated CNVs using qPCR.
No evidence was found that fusion-gene generating CNVs led to ASD susceptibility. However, the discovery of a MAPKAPK5-ACAD10 transcript with an estimated frequency of ~1/200 suggests that gain-of-function mechanisms should be considered in future studies of genomic imbalance and disease susceptibility.
Holt, R., et al. Eur. J. Hum. Gen. doi: 10.1038/ejhg.2012.73 (2012) - CNVs Leading to Fusion Transcripts in Individuals with Autism Spectrum Disorder