Successfully develop a PCR protocol to detect low copy DNA

I am interested in studying post-transcriptional regulatory mechanisms that occur during tumorigenesis. One of the major technical challenges of my project is being able to determine expression of exogenously introduced mutated genes in a sample. Developing a PCR assay to distinguish the mutated genes from the endogenous wild type copy proved to be virtually impossible, as I could not detect any mutant transgenes in my samples. This set back my research dramatically, and I had very few options in re-designing primers since there are very few sequence differences between the mutant and wild type genes. To make matters worse, the region I was forced to design primers to was extremely GC rich. Fortunately, a colleague introduced me to MyFi DNA polymerase. Using this product I was able to successfully detect my mutant gene of interest. My gene of interest was expressed in my samples but it was a very low percentage of total DNA. MyFi allowed me to successfully develop a PCR protocol to detect low copy DNA when other PCR products, including traditional PCR and quantitative real time PCR, did not.
Laura Simone
Duke University
Molecular Genetics & Microbiology

Bioline Scholar: Focus on Autism Spectrum Disorders

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.

MyTaq Red Mix
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

BIO-X-ACT Long DNA Polymerase
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

Bioline Scholar: Focus on MRSA

The groundbreaking discovery by Sir Alexander Fleming of the first antibiotic penicillin in 1929, and its subsequent isolation by Ernst Chain and Sir Howard Florey in 1939, led to the award of the Nobel prize in 1945. This discovery revolutionized modern medicine, paving the way for the development of many more natural antibiotics such as chloramphenicol, streptomycin and tetracycline. These antibiotics were effective against the full array of bacterial pathogens including Gram-positive and Gram-negative bacteria, intracellular parasites and Mycobacterium tuberculosis.

However, over the past several decades, bacteria have developed resistance to existing drugs. Drug resistance by so-called “superbugs” is a severe public health problem, whilst development of new generation anti-microbials faces great challenges.

Earlier this year, the WHO released a sobering update on drug-resistant TB; it was reported to be at the highest rates ever recorded.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important nosocomial pathogens. For example, an estimated 89,785 invasive MRSA infections were associated with 15,249 deaths in 2008 in the United States. Even when the infection is successfully treated it can double the average length of a hospital stay and thereby increase healthcare costs. MRSA is able to produce a range of unique toxins causing severe infections such as endocarditis, osteomyelitis, pneumonia and septic shock in patients with open wounds, invasive devices and weakened immune systems.

One part of the problem is that bacteria and other microbial pathogens are very hardy and have developed different antimicrobial resistance mechanisms. Several interesting acronyms have been coined to describe the range of resistance from multi-drug resistant (MDR), extensively drug resistant (XDR), to totally drug resistant (TDR). The increasing use and misuse of existing antibiotics in human and veterinary medicine as well as agriculture has also exacerbated the problem. Antimicrobial resistance is genetically based; resistance is mediated by the acquisition of extrachromosomal genetic elements containing resistance genes, such as plasmids, transposable genetic elements and genomic islands, which are transferred between bacteria via horizontal gene transfer.

Last month, a new study published in the New England Journal of Medicine was able to identify which isolates of MRSA were part of a hospital outbreak using whole genome sequencing. Scientists from the Wellcome Trust Sanger Institute, University of Cambridge and Illumina sequenced the isolates within a timescale that could influence infection control and patient management. The authors created an artificial “resistome” of antibiotic-resistance genes and demonstrated concordance between it and the results of phenotypic susceptibility testing. They also created a “toxome” consisting of toxin genes.

Bioline offers a range products for use in microbial research and below we document some of the publications in which Bioline reagents were used to advance research.

ImmoMix
The Petez-Osorio group used ImmoMix Red to describe a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampinr), isoniazidr, and pyrazinamider TB. This MID-DRS assay reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as two days with reduced assay costs, preparation time and risks due to user errors.

Petez-Osorio, A. C., et al. J. Clin. Microbiol. 50(2): 326-336 (2012) Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing

SensiMix SYBR One-Step Kit
The Rosato group at the Center for Molecular and Translational Human Infectious Diseases Research, Methodist Hospital Research Institute in Houston, USA conducts translational research focusing on multidrug-resistant bacterial pathogens. The aim of their study was to identify the molecular genetic causes of drug resistance and resistance gene spread in MRSA.

In this paper, Dr Rosato’s group used DNA microarrays and qRT-PCR to evaluate differential gene expression during HeR-HoR selection and found increased expression of the agr two-component regulatory system. Their findings reinforce the concept that increased expression of agr during HeR-HoR selection plays a critical role in regulating the ß-lactam-induced increased mutation rate in very heterogeneous MRSA strains.

Plata, K. B., et al. Antimicrob. Agents Chemother. 55(7): 3176-3186 (2011) Fate of Mutation Rate Depends on agr Locus Expression during Oxacillin-Mediated Heterogeneous-Homogeneous Selection in Methicillin-Resistant Staphylococcus aureus Clinical Strains

In another exciting paper from Dr Rosato’s group at the Methodist Hospital Research Institute, Houston, they teamed up with researchers from Virginia Commonwealth University and Cubist Pharmaceuticals to investigate the molecular basis of resistance to daptomycin (DAP), a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed drug mechanisms include disruption of the bacterial membrane wall.

Differential gene expression analysis using SensiMix One-Step Kit also showed up of the two-component regulatory system vraSR. Crucially, this effect was related to the impact of vraSR and mprF mutations in the cell wall. Their work underscores the suggestion that alterations in these two genes contributes to DAP-resistance in this group of clinical MRSA strains.

Mehta, S., et al. Antimicrob. Agents Chemother. 56(1): 92-102 (2012) VraSR Two-Component Regulatory System Contributes to mprF-Mediated Decreased Susceptibility to Daptomycin in In Vivo-Selected Clinical Strains of Methicillin-ResistantStaphylococcus aureus

BIO-X-ACT DNA Polymerase
The Morrissey group at the University of Leicester, UK conducts research into microbial genetics. One of their major areas of investigation is the Staphylococcus aureus adaptive response to antibacterial copper. Their results shows copper resistance varies considerably between clinical strains due to the carriage of an additional plasmid-encoded copper homeostasis mechanism, copBmco. Importantly, this plasmid has the potential to spread to other S. aureus strains. This is the first time that plasmid-encoded copper resistance has been reported and shown to be transferable between pathogenic bacteria isolated from humans.

Baker, J., et al. Environ. Microbiol. 13(9): 2495-2507 (2011) The Staphylococcus aureus CsoR regulates both chromosomal and plasmid-encoded copper resistance mechanisms

MangoTaq DNA Polymerase
The aim of this study at St. Vincent’s Hospital in Australia was to determine the utility of molecular methods compared to selective agars for MRSA detection. The conclusion of the van Hal group was that molecular detection methods for MRSA remain sensitive and rapid, but are associated with greater expense.

van Hal, S. J., et al. Euro. J. Clin. Microbiol. & Infectious Dis. 28(1): 47-53 (2010) MRSA detection: comparison of two molecular methods (BD GeneOhm PCR assay and Easy -Plex) with two selective MRSA agars (MRSA-ID and Oxoid MRSA) for nasal swabs

Bioline Scholar: Plant Epigenetics

Focus on Plant Epigenetics

2012 marks the 150th anniversary of the publication of Charles Darwin’s first botanical book, on the fertilization of orchids (1862), wherein he described pollen grains and outlined his evolutionary principles with respect to plant research. Gregor Mendel was also coming to the end of this famous pea study (1856-1863). The ‘green biotech-revolution’ of the 21st century can be traced back to these classical breeding experiments; however, with the use of molecular biology – and in particular the information we have gained from the sequencing of many plant species – there has been an unprecedented development in every field of plant science in the last few years.

Plants are crucial to human society as they provide everything from habitat to other essentials for survival such as food, oxygen, and medicines, while also creating and preserving the essential soils required for this cycle to continue. Plants are also required for other, non-essential items such as cosmetics and an almost infinite range of products for people and society. As human population increases, so too does the our dependence on plants and, consequently, the need to understand plants and their potential in the coming decades is increasing, leading to greater emphasis on the calls for basic plant biological research.

Bioline offers a number of reagents that have helped further the study and understanding of plants. This month’s edition of Bioline Scholar focuses on the use of Bioline reagents and kits in the field of plant epigenetic research.

SensiFAST™ SYBR & Fluorescein Kit
The results in this exciting paper from the Gregor Mendel Institute of Molecular Plant Biology in Austria suggest that DMS11 (defective in meristem silencing 11) provides the missing ATPase function for DMS3 and that these proteins cooperate in the RNA-directed DNA methylation pathway to promote transcriptional repression. GHKL ATPases are thus emerging as new players in epigenetic regulation in plants and mammals.

Lorkovic C., et al. Current Biol. 22: 1–6 (2012) – Involvement of a GHKL ATPase in RNA-Directed DNA Methylation in Arabidopsis thaliana [PDF]

SensiMix™ SYBR No-ROX Kit
Jones and colleagues studied the flowering of Eucalyptus globulus subsp. globulus, an important forestry species in temperate parts of the world. The globulus homologues, FLOWERING LOCUS T (FT) and LEAFY (LFY) were examined shown to form part of the flower initiation pathway in Eucalyptus but do not regulate the observed differences in anthesis time.

Jones R. C., et al. Australian J. Botany 59 (8): 756-769 (2012) – Expression of a FLOWERING LOCUS T homologue is temporally associated with annual flower bud initiation in Eucalyptus globulus subsp. globulus (Myrtaceae)

hmdCTP (5-hydroxymethyl-2′-deoxycytidine 5′-triphosphate)
Yao’s study asks how well the Arabidopsis thaliana Variant in Methylation 1 (VIM1) protein, an essential factor in maintaining 5-cytosine methylation (5mC) homeostasis and epigenetic silencing in this plant, recognizes 5-hydroxymethyl-cytosine (5hmC). They found that 5hmC may contribute to VIM-mediated passive loss of cytosine methylation in vivo during Arabidopsis DNA replication.

Yao Q., et al. Protein Expression and Purification 83 (1): 104–111 (2012) – Heterologous expression and purification of Arabidopsis thaliana VIM1 protein: In vitro evidence for its inability to recognize hydroxymethylcytosine, a rare base in Arabidopsis DNA

MangoTaq™ DNA Polymerase
The characteristic expression pattern of AtBMI1C is the result of a complex epigenetic process. The results show the orchestrated interplay of different epigenetic mechanisms in regulating gene expression throughout development, shedding light on the current hypotheses for the origin and mechanism of imprinting in plant endosperm.

Bratzel, F., et al. Mol. Plant 5 (1): 260-269 (2012) – Regulation of the new Arabidopsis imprinted gene AtBMI1C requires the interplay of different epigenetic mechanisms

BioMix™
A targeted epigenetic response to a specific environmental stress in a physiologically important pathway is reported, suggesting epigenetic regulation of stomatal development that allows for anatomical and phenotypic plasticity, and may help to explain at least some of the resilience to fluctuating relative humidity.

Tricker P. J., et al. J. Expt. Botany doi: 10.1093/jxb/ers076 (2012) – Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development.

BIO-X-ACT™ Short DNA Polymerase
Next-generation sequencing of barcoded amplicon mixtures was used to reliably sample all alleles of homeologous loci in polyploid species and successfully investigate phylogenetic relationships among species, as well as to investigate phylogeographic hypotheses. This next-generation sequencing method is more affordable than and at least as reliable as bacterial cloning. It could be applied to any experiment involving sequencing of amplicon mixtures.

Griffin P. C., et al. BMC Biol. 9: 19 (2011) – A next-generation sequencing method for overcoming the multiple gene copy problem in polyploid phylogenetics, applied to Poa grasses

In Arabidopsis thaliana, the ability to flower is mainly related to a floral repressor, FLOWERING LOCUS C (FLC), which is regulated through the vernalization pathway. This paper shows that the regulation of CiMFL gene expression in time and space and in relation to environmental conditions is only partially conserved with respect to FLC in A. thaliana. A model for flowering repression by CiMFL is proposed.

Locascio A., et al. New Phytologist 182 (3): 630–643 (2009) – Characterization of a MADS FLOWERING LOCUS C-LIKE (MFL) sequence in Cichorium intybus: a comparative study of CiMFL and AtFLC reveals homologies and divergences in gene function

BioMix™
The most economically important species is O. ficus indica, cultivated both for fruits and cladodes, but little is known about their ancestries and levels of genetic diversity. This study increases our knowledge of the variability among some of the most diffused Opuntia cultivated accessions and also points to the inconsistencies of previous taxonomical genotype assignments that were based solely on morphological characteristics.

Caruso M., et al. Plant Syst. Evol. 290 (1-4): 85-97 (2010) – Microsatellite markers help to assess genetic diversity among Opuntia ficus indica cultivated genotypes and their relation with related species

Bioline Scholar Monthly: April 2012 Roundup – Breast Cancer

Focus on Breast Cancer

For years scientists have been treating breast cancer as a single disease. However, a new landmark study published in Nature has reclassified breast cancer into ten separate sub-diseases based on their genetic fingerprint. The culmination of decades of research, the study is the largest global study of breast cancer tissue ever performed.

The team, led by the British Columbia Cancer Center in Canada and the Cambridge Cancer Research Institute in the UK, used genome-wide microarrays to analyze the DNA and RNA of 2,000 tumor samples taken from women diagnosed with breast cancer. This huge pool of genetic information (copy number variants, SNPs and gene expression data), as well as survival data, allowed researchers to spot new and previously unacknowledged patterns for ten subtly different cancers that have, historically, been considered as one.

The challenge now is to understand the genetic drivers behind these newly discovered breast cancer variants and to develop new targeted therapies in the future. It could also lead to women with the best prognosis being spared side-effects of chemotherapy. The classification system will likely also form the basis for newer and better ways to diagnose and manage the disease.

Bioline offers a number of reagents that have helped further the study of cancers and, more specifically, breast cancer. So this edition of Bioline Scholar Monthly focuses on the use of Bioline reagents and kits in the field of breast cancer research.

SensiMix SYBR & Fluorescein Kit
In a diverse cohort of breast cancer patients with a 1–5 year tumor relapse versus those with up to 7 years relapse-free survival, RNA was extracted and subjected to microarray and real-time RT-PCR analysis. Among the 299 genes, five genes which included B cell response genes were found to predict with >85% accuracy relapse-free survival. Real-time RT-PCR confirmed the 5-gene prognostic signature that was distinct from an FDA-cleared 70-gene signature of MammaPrint panel and from the Oncotype DX recurrence score assay panel.

Ascierto, L. M., et al. Breast Can. Res. Treat. 131(3):871-880 (2012) – A signature of immune function genes associated with recurrence-free survival in breast cancer patients.

TRIsure reagent
MicroRNAs (miRNAs) are noncoding RNAs that function as key posttranscriptional regulators of gene expression. This paper found that BRCA1 recognizes the RNA secondary structure and directly binds with primary transcripts of miRNAs via a DNA-binding domain. The findings indicate novel functions of BRCA1 in miRNA biogenesis, which may be linked to its tumor suppressor mechanism and maintenance of genomic stability.

Kawai S. and Amano A. J. Cell Biol. 197 (2):201-208 (2012) – BRCA1 regulates microRNA biogenesis via the DROSHA microprocessor complex.

SensiMix SYBR No-ROX Kit
CD44, the transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. The expression of CD44 and its variants is associated with poor prognosis in breast cancer. This paper investigated the effect of silibinin (a polyphenolic flavonolignan of the herbal plant of Silybum marianum, milk thistle) on the epidermal growth factor (EGF) ligand-induced CD44 expression in human breast cancer cells. The results suggest that silibinin prevents the EGFR signaling pathway and may be used as an effective drug for the inhibition of metastasis of human breast cancer.

Kim S., et al. Anticancer Res. 31(11): 3767-3773 (2011) – Silibinin Suppresses EGFR Ligand-induced CD44 Expression through Inhibition of EGFR Activity in Breast Cancer Cells.

Human papillomavirus (HPV) and Epstein Barr virus (EBV) have been found in breast carcinomas around the world. In this study, fifty-five BCs from Chile were analyzed for HPV and EBV presence. In addition, HPV- 16 viral load/physical status and E6/E7 expressions were determined. The results suggest that it is unlikely that HPV and/or EBV play a direct role in the etiology of breast carcinomas.

Aguayo F., et al. Infectious Agents and Cancer 6:7 (2011) – Human papillomavirus and Epstein-Barr virus infections in breast cancer from chile.

cDNA Synthesis Kit
This study suggests that melatonin may play a role in the desmoplastic reaction in breast cancer through a down regulatory action on the expression of antiadipogenic cytokines, which decrease the levels of these cytokines. Lower levels of cytokines stimulate the differentiation of fibroblasts and decrease both aromatase activity and expression, thereby reducing the number of estrogen-producing cells proximal to malignant cells.

Alonso-González C., et al. J. Pineal Res. 52(3): 282–290, (2012) – Melatonin interferes in the desmoplastic reaction in breast cancer by regulating cytokine production.

Melatonin reduces the development of breast cancer interfering with oestrogen-signalling pathways, and also inhibits aromatase activity and expression. This study shows that melatonin inhibits aromatase activity and expression by regulating the gene expression of specific aromatase promoter regions. A possible mechanism for these effects would be the regulation by melatonin of intracellular cAMP levels, mediated by an inhibition of cyclooxygenase activity and expression.

Martínez-Campa C., et al. British J. Can. 101: 1613–1619 (2009) – Melatonin inhibits aromatase promoter expression by regulating cyclooxygenases expression and activity in breast cancer cells.

IMMOLASE DNA Polymerase
Bisphenol A (BPA) has long been suspected to promote carcinogenesis, but the high doses of BPA used in many studies generated conflicting results. This paper shows that BPA at environmentally relevant doses reduces the efficacy of chemotherapeutic agents. These data provide considerable support to the accumulating evidence that BPA is hazardous to human health.

LaPensee E. W., et al. Environ Health Perspect. 117(2): 175–180 (2009) – Bisphenol A at Low Nanomolar Doses Confers Chemoresistance in Estrogen Receptor-a–Positive and –Negative Breast Cancer Cells.

Bioline Celebrates 20 Years of Service to Life Science

How time flies! It’s 2012 and this year Bioline: The PCR Company celebrates its 20 year anniversary.

The world has changed rapidly and dramatically since Bioline was first established in 1992 by Marco Calzavara, president of Bioline, but we are still manufacturing and supplying a range of consistently high-quality products to life science researchers and institutions around the world.

From our base in London, UK, we have rapidly expanded to become a leading primary manufacturer and distributor of PCR enzymes and reagents for molecular biology; providing complete solutions to researchers in universities, major research institutions, hospitals, biotech firms, diagnostics and pharmaceutical firms and laboratories globally. Bioline’s products have been cited thousands of times in leading journals by researchers all over the world.

When asked about the milestone of two decades of service provision to life science, Marco Calzavara, founder and president of Bioline, commented:

“Our aim at Bioline for the past 20 years has been to develop high-quality and reliable products that really make the difference, delivering consistent results time after time. I am excited and proud to continue Bioline’s great work as a cutting-edge molecular biology company, ensuring that we maintain our outstanding reputation as a high-quality and reliable firm.”

Richard L. Eberly, chief commercial officer of Meridian Bioscience, Inc., added:

“Congratulations to the Bioline team on its 20th anniversary of successful operations throughout the world. We are enthusiastic about the success of the merger and impressed by the scientific capabilities of Bioline, as well as its well-deserved reputation for innovation and quality. We remain committed to expanding the rapidly growing portfolio of highly-specialized molecular biology products from Bioline that enable the development of genomic tests utilized by researchers, clinical diagnostic laboratories, diagnostic test manufacturers and biotechnology companies.”

As part of the 20 year celebrations, we are running a monthly Mystery Prize Giveaway for the rest of 2012. Lucky customers will find a golden ticket hiding in their delivery, so keep a close eye out when you receive your delivery. You may find one included with your next order!

We’d like all our customers over the years to join in the celebration. You can do so by visiting the special Bioline 20 Year Anniversary page, where we have a timeline listing the major milestones over the last 20 years and an exclusive interview with our president, as well as other Bioline employees. You can also keep up to date with all the latest 20 year anniversary news and updates by following @BiolineUSA on Twitter and by joining us on Facebook.

Thank you to all of our customers, both old and new alike. We are looking forward to supplying you with more high-quality enzymes and reagents for the next 20 years!

How to Take Your PCR From 11 Hours to TWO

Fides Lay

We caught Fides Lay in the lab on the day of a basketball game between UCLA and crosstown arch-rival USC. As a former UCLA undergrad now working on her PhD at USC, she never wants to miss a game between the two. Unfortunately, the long hours in the lab don’t always allow her to be home in time to watch. At least, that’s how it used to be for her. Thanks to Bioline’s MyTaq HS Mix, now she doesn’t miss a game.

In Peter Jones’ lab at USC, Fides is part of a team studying the epigenetic regulation of cancer. And as anyone who ever worked on epigenetics knows, you need to be a master of PCR to get any results at all. When you need to amplify bisulfite converted DNA to measure methylation status, you work with small samples that often have been digested with several enzymes, with DNA that is fragmented and damaged.

For the longest time the lab amplified bisulfite converted DNA with a tedious protocol. Tedious because of the long set-up with GoTaq®, DMSO and other components added one at a time, and extremely slow cycling with a PCR protocol that was 7-11 hours long. Anything could go wrong at any time, and the result was impossible to predict. Simply using a different thermocycler with a different ramping speed could cause the reaction to fail. And once you found out, it would be too late to fix because every new attempt takes a whole other day. Since the lab usually clones and sequences the PCR fragments, there was always still the risk that no clones would have inserts. Hard to predict, and that meant starting over.

All that changed when Fides first tried Bioline’s MyTaq HS Mix, an easy, all-in-one mix that contains the enzyme, dNTPs, buffer and all optimizers. There’s no need to add any DMSO, it works right away on almost all templates. It’s fast. Reactions are done in less than two hours even on bisulfite converted DNA, with very consistent results and always with nice bands. The PCR products are much easier to clone, and on the rare occasion that something does go wrong, there’s still time to redo the experiment AND get the samples off to pyrosequencing the same day.

Now Fides has time to run multiple experiments and redo anything that goes wrong, all in time to get home, kick up her feet and watch the game.