Monthly Archives: July 2017

Things are about to speed up dramatically in genetic research, with scientists developing a new technique that can clone thousands of genes in a single reaction.

The new technology, called a LASSO probe, could be used to create libraries of proteins from DNA samples, speeding up the search for new drugs by replacing the tedious methods of gene cloning currently used.

When you think of cloning you may think of Dolly the sheep or the company that promises to clone your favourite pet so you don't have to live sad and alone, but that's a different kind of cloning. Here we're talking about molecular cloning, a natural process that occurs when bacteria, insects, or plants reproduce without a partner.

Scientists clone DNA because they want to do one of two things; either they want to gain information about a particular gene or they want to manipulate genetic information in a cell to give the cell a new property. Both reasons require scientists to have millions of copies of the same DNA molecule in a test tube.

At the moment, to work out what a gene does by cloning its DNA and expressing its protein is done one gene at a time. The standard sequencing method, called molecular inversion probes, involves capturing small fragments of DNA (about 200 base pairs long) and connecting them together to map out the full genome code.

Weaving together these small sections of code to form the full gene sequence isn't easy, but there hasn't been any other way to sequence long fragments of DNA and it's been holding research back.

Not to scale. Credit: Jennifer E. Fairman/Johns Hopkins University

"We think that the rapid, affordable, and high-throughput cloning of proteins and other genetic elements will greatly accelerate biological research to discover functions of molecules encoded by genomes and match the pace at which new genome sequencing data is coming out," says one of the team, Biju Parekkadan, from Rutgers University.

In this new study, the LASSO probe - which stands for "long adaptor single stranded oligonucleotide" - can capture and clone thousands of long DNA fragments and the researchers hope that the new technique will push the limits of what we can currently do.

"Our goal is to make it cheap and easy for any researcher in any field to clone and express the entire set of proteins from any organism," said co-researcher Ben Larman from Johns Hopkins University School of Medicine. "Until now, such a prospect was only realistic for high-powered research consortia studying model organisms like fruit flies or mice."

How does this new technique work?

A collection of LASSO probes were used to grab desired DNA sequences, you can think of it like the way a rope lasso is used to capture cattle. Instead of aiming for the spiky horns of a cow, the LASSO probe targets a DNA sequence up to a few thousand base pairs long - the typical length of a gene's protein code.

The study is a proof of concept, with the LASSO probes used to capture over 3,000 DNA fragments from the E. coli bacterial genome. The results show the probes successfully captured around 75 percent of the gene they targeted.

There were also other benefits to the LASSO probe technique.

The researchers say that the sequences are captured in a way that allows them to also analyse what the genes' proteins do and demonstrated this by giving antibiotic resistance to a cell that would otherwise be killed by the antibiotic.

The researchers were also able to capture and clone a protein library from a human microbiome sample and they hope that it will lead to improved precision medicine and rapid discovery of new medicines for a range of diseases.

"We're very excited about all the potential applications for LASSO cloning," said Larman. "Our hope is that by greatly expanding the number of proteins that can be expressed and screened in parallel, the road to interesting biology and new therapeutic biomolecules will be dramatically shortened for many researchers."

The study has been published in Nature Biomedical Engineering.

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Scientists Can Now Clone Thousands of Genes in a Single Reaction - ScienceAlert

Radiation therapy (RT) remains a mainstay of modern oncologic treatment, with more than half of all patients receiving RT during their treatment course. However, individual responses to RT vary widely among disease types and patient populations.[1] Recent years have been marked by the development and expanded use of precision medicine in cancer therapeutics. Precision medicine refers to the tailoring of treatment to the individual characteristics of each patient, based on inherent susceptibilities. Although enormous strides have been made in tailoring a variety of approaches to systemic therapy, the role of radiation oncology in precision medicine is just beginning to emerge.[1]

Precision in RT has been advancing along multiple parallel paths. There have been improvements in the precision of anatomic target delineation with the use of intensity-modulated RT, volumetric arc therapy, and stereotactic RT, all of which allow for improved target dose conformality. Concurrent with technical advances in treatment delivery, the field of radiogenomics, or the interplay between genomic elements and radiation response at the cellular level, continues to evolve. Indexing the determinants of radiation response at the cellular level has the potential to allow for more personalized delivery of RT and to further increase the therapeutic ratio of our treatment.[1]

As the rates of cancer survival continue to improve, the effect of treatment toxicity on normal tissue will play an increasingly important role in treatment selection. Capturing patient-reported outcomes from the growing and evolving survivor population sheds light on the potential far-reaching impact of radiogenomics beyond traditional survival measures. Specifically, by recognizing the connection between genotypic variation and normal tissue response, our ability to predict severe toxicities following RT may spare selected individuals from significant morbidity and mortality following treatment.[2] Moreover, studies investigating genetic assays predictive of tumor radiosensitivity may be complementary to studies evaluating the radiosensitivity of noncancerous tissue.[3] The purpose of the current article is multifold: Herein, we will review the background and history of genomic predictors of RT response; evaluate candidate genes and polymorphisms dictating responses to radiation; discuss emerging data on the use of genetic signatures; and review current guidelines on the use of genomic predictors to tailor therapy. The article is structured to discuss outcomes and toxicities based on precision medicine in RT within each of these sections.

Biomarkers have long been used in the field of oncology as an adjunct to traditional staging information to estimate treatment outcomes. In this field of study, it is important to distinguish between prognostic and predictive biomarkers. Prognostic markers are associated with a clinical outcome, such as overall survival (OS), regardless of the treatment delivered.[4] For example, the prostate-specific antigen (PSA) has been proven to be an important biomarker in prostate cancer, correlating with the risk of recurrence and OS. Although elevated PSA levels are associated with worse outcomes, measurement of PSA alone does not yet predict the patient response to specific treatments.

Predictive markers, on the other hand, are indicators of the likely benefit following specific treatment. These markers are therefore useful in tailoring treatment decisions. An example of a predictive marker is ERBB2 gene amplification (resulting in overexpression of human epidermal growth factor receptor 2 [HER2]) in breast cancer, since clinical outcomes are improved by the addition of trastuzumab to the chemotherapy regimen in patients with this genetic aberration.[4] The National Comprehensive Cancer Network (NCCN), in updating the NCCN Biomarkers Compendium, recently released a task force report addressing the use of molecular biomarkers in six major disease sites.[5] While prognostic biomarkers provide important information regarding clinical outcome, implicit to the goal of precision medicine is the identification of predictive biomarkers to help direct individual treatment. Despite the significant progress made by radiogenomics in this regard over the past 20 yearsfrom focused gene studies to genome-wide association studies (GWAS)in the field of radiation oncology, clinical translation of these principles remains a goal on the horizon.[2]

Studies investigating variable responses of tissues to RT date back more than 60 years ago to the investigations carried out by Gray and colleagues.[6-8] Specifically studied was the effect of oxygenation on RT response. The tumor microenvironment has been demonstrated to have topographic variability; certain regions possess particularly low extracellular pH, low nutrient content, and hypoxia. Given the often tortuous and malformed vasculature of tumors, blood flow to the microenvironment contributes to an imbalance in the supply of and demand for oxygen. The resulting hypoxia correlates with tumor cell radioresistance, since the maximal effect of RT is achieved by the generation of free radicals.[6] Preceding the early discovery of the effect of hypoxia on radioresistance was the demonstration of individual variation in the response of normal tissue following treatment with a given dose of radiation. This was first formally described in 1936 with the publication of the now well-described sigmoid doseresponse curve.[9] Alongside the discovery of differing individual responses to similar radiation doses was the detection of RT hypersensitivity in patients with certain rare genetic syndromes. The first such documented adverse reaction occurred in a 10-year-old patient with mutation of the ATM gene, who died from complications related to radiation toxicity in normal tissues. Since this initial case was reported, the ATM mutation has been intricately linked with the DNA damage response and studied extensively.[10] While the demonstration of radiosensitivity in patients with rare genetic disorders has been instrumental in our understanding of differential radiation responses, it does not yet explain the wide range of radiation responses seen in patients without known genetic syndromes.

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The Role of Genomic Techniques in Predicting Response to Radiation Therapy - Cancer Network