This blog was written by guest blogger and 2018 Promega Social Media Intern Logan Godfrey.
Only 30 years ago, the polymerase chain reaction (PCR)
was used for the first time, allowing the exponential amplification of a specific
DNA segment. A small amount of DNA could now be replicated until there was
enough of it to study accurately, even allowing sequencing of the amplified DNA.
This was a massive breakthrough that produced immediate effects in the fields
of forensics and life science research. Since these technologies were first
introduced however, the molecular biology research laboratory has been the sole
domain of PCR and DNA sequencing.
While an amazing revolution, application of a technology
such as DNA sequencing is limited by the size and cost of DNA sequencers, which
in turn restricts accessibility. However, recent breakthroughs are allowing DNA
sequencing to take place in jungles, the arctic, and even space—giving science
the opportunity to reach further, faster than ever before.
The newfound accessibility of DNA sequencing means a
marriage between fields of science that were previously largely unacquainted.
The disciplines of genomics and wildlife biology/ecology have largely progressed
independently. Wildlife biology is practiced in the field through observations
and macro-level assessments, and genomics, largely, has developed in a lab
setting. Leading the charge in the convergence of wildlife biology and genomics
is Field Projects International.
Knowing how much DNA you have is fundamental to successful experiments. Without a firm number in which you are confident, the DNA input for subsequent experiments can lead you astray. Below are six reasons why you should quantitate your DNA.
6. Saving time by knowing what you have rather than repeating experiments. If you don’t quantitate your DNA, how certain can you be that the same amount of DNA is consistently added? Always using the same volume for every experiment does not guarantee the same DNA amount goes into the assay. Each time there is a new purified DNA sample, the chances that you have the same quantity as before are lessened. Consequently, without knowing the DNA concentration of the sample you are using, the amount of input DNA cannot be guaranteed and experiments may have to be repeated.
Next-generation sequencing (NGS), also known as high-throughput parallel sequencing, is the all-encompassing term used to describe a number of different modern sequencing technologies. These include Illumina (Solexa) sequencing, Roche 454 sequencing, Ion torrent: Proton / PGM sequencing and SOLiD sequencing to name a few .
With the advent of these technologies sequencing DNA and RNA has become much more facile and affordable in comparison to the previously used Sanger sequencing. For these reasons NGS has been the game-changer in the field of modern genomics and molecular biology.
A common starting point for template preparation for NGS platforms is random fragmentation of target DNA and addition of platform-specific adapter sequences to flanking ends. Protocols typically use sonication to shear input DNA, coupled with several rounds of enzymatic modification to produce a sequencer-ready product .
Accurate quantification of DNA preparations is essential to ensure high-quality reads and efficient generation of data. Too much DNA can lead to issues such as mixed signals, un-resolvable data and lower number of single reads. Too little DNA, on the other hand, might result in insufficient sequencing coverage, reduced read depth or empty runs, all of which would incur higher costs. The quality of DNA can also vary depending on the source or extraction method applied and further reinforces the need for appropriate management of the input material. Continue reading “Simplifying Next Generation Sequencing Workflow with QuantiFluor® ds-DNA System”
For most molecular biology applications, knowing the amount of nucleic acid present in your purified sample is important. However, one quantitation method might serve better than another, depending on your situation, or you may need to weigh the benefits of a second method to assess the information from the first. Our webinar “To NanoDrop® or Not to NanoDrop®: Choosing the Most Appropriate Method for Nucleic Acid Quantitation” given by Doug Wieczorek, one of our Applications Scientists, discussed three methods for quantitating nucleic acid and outlined their strengths and weaknesses. Continue reading “Methods for Quantitating Your Nucleic Acid Sample”
Yesterday I listened in on the Webinar “Getting the Most Out of Your DNA Analysis from Purification to Downstream Assays”, presented by Eric Vincent–a Product Manager in the Promega Genomics group.
This is the webinar for you if you have ever wondered about the relative advantages and disadvantages of the many methods available for DNA purification, quantitation and analysis, or if you are comparing options for low- to high-throughput DNA purification. Eric presents a clear analyses of each of the steps in a basic DNA workflow: Purification, Quantitation, Quality Determination, and Downstream Analysis, providing key considerations and detailing the potential limitations of the methods commonly used at each step.
The DNA purification method chosen has an affect on the quality and integrity of the DNA isolated, and can therefore affect performance in downstream assays. Accuracy of quantitation also affects success, and the various downstream assays themselves (such as end-point PCR, qPCR, and sequencing) each have different sensitivities to factors such as DNA yield, quality, and integrity, and the presence of inhibitors. Continue reading “DNA Purification, Quantitation and Analysis Explained”
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