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NEXTGENPCR Amplifies the Entire BRCA1 Gene in 10 Minutes [POSTER] | 16 JUL 2018

With optimization and the use of a fast enzyme, the NEXTGENPCR thermocycler amplified a 100 bp fragment in less than 2 minutes. But what about larger fragments?

A poster presented at the European Society of Human Genetics 2016 Annual Meeting demonstrated how the NEXTGENPCR thermocycler amplified the entire BRCA1 gene within 10 minutes using 29 primer pairs. The PCR was followed by Sanger sequencing.
 

How it was done:

 

The BRCA1 gene fragments were amplified under these conditions.

 

Initial denaturation took place at 98°C for 60 seconds.

29 cycles total:

  • Denaturation for 3 seconds at 98°C
  • Annealing for 7 seconds at 60°C
  • Extension for 6 seconds at 75°C        

NEXTGENPCR is redefining fast. The NEXTGENPCR thermocycler heats and cools samples instantly, losing no time in getting samples to the desired temperature. Go from melting to annealing in less than 0.1 seconds. Download the poster content below to get all the experimental details.

 

 

DOWNLOAD THE POSTER 

 

 

Products mentioned are for Research Use Only.  Not for use in diagnostic procedures.
Canon BioMedical is the exclusive distributor of NEXTGENPCR in the USA.
The NEXTGENPCR products are manufactured by Molecular Biology Systems, B.V.
All referenced product names, and other marks, are trademarks of their respective owners
Nothing herein constitutes medical advice.

PCR in 1 Minute and 59 Seconds – How It Was Done | 09 JUL 2018

DNA amplification using PCR is a necessary step in most molecular biology procedures. Molecular Biology Systems (MBS) developed NEXTGENPCR, a novel thermocycler that uses distinct thermal zones to denature, anneal and extend, eliminating ramp times. The thermal zones are maintained at a set temperature, and the samples move between thermal zones so that only the sample temperature changes. The unique design of the sample plates along with the heat blocks in each thermal zone makes the sample temperature change instantaneous. See our blog post NEXTGENPCR – Discover How It Works from June 4 for more details. By optimizing the protocol using a fast polymerase with NEXTGENPCR, a 100 bp fragment was amplified in just 1 minute and 59 seconds. 

 

How it was done

Figure 1. PCR product of a 100 bp fragment compared to a 100 bp ladder, visualized on 2% agarose

 

  1. Samples were heated to 98°C for 10 seconds
  2. Five cycles with the following settings:
    • Denaturation at 98°C for 2 seconds 
    • Annealing at 60°C for 2 seconds
    • Extension at 75°C for 2 seconds
  3. Twenty-five cycles with the following settings:
    • Denaturation at 98°C for 0.5 seconds
    • Annealing at 60°C for 0.5 seconds
    • Extension at 75°C for 0.5 seconds

 

With the elimination of ramp times and a bit of optimization, it is possible to cut your
time to results. Download the application note to read the full experiment.

 

 

DOWNLOAD NOW ;

 

 

Canon BioMedical is the exclusive distributor of NEXTGENPCR in the United States.
Products mentioned are for Research Use Only.  Not for use in diagnostic procedures.
The NEXTGENPCR products are manufactured by Molecular Biology Systems, B.V.
Experiment results are for informational purposes only.
All referenced product names, and other marks, are trademarks of their respective owner.

 

 

Ten Tips for Preventing Amplicon Contamination | 02 JUL 2018

Amplicon contamination poses a real problem for laboratories performing PCR-based experiments. Amplicon contaminants can come from any RNA or DNA, from a previous PCR amplification, or from a laboratory procedure, such as a plasmid preparation or in vitro transcription. If proper precautions are not taken to prevent amplicon contamination, the contaminants can serve as a template and interfere with your experiments by creating false positives or other undesirable results.

Here are 10 tips for preventing amplicon contamination in your lab:

  1. Define a lab workflow. The lab workflow will look different depending on your needs and resources. If you have space, set up your PCR in a separate room from both general lab procedures and downstream processing of amplicon-generating PCR experiments. Otherwise, establish a physical workflow within the same room by denoting specific areas with signage or tape markings. Any level of one-way flow that you establish, thus eliminating backflow, will help prevent contamination.

  2. Color code different zones of your lab. Put those multicolored tube racks to good use by organizing them around your established workflow. For example, use green for pre-PCR work, yellow for PCR setup and run, and red for post-PCR processing and analysis. Follow the same color scheme throughout, wherever possible, using tape, pipette labels, racks, etc.  Use a different labcoat, fresh gloves, and safety glasses, each stored in the respective zone to help prevent carryover. Isolating anything that is touched during lab work, to a given zone, will limit contamination.

Tips for Preventing Amplicon Contamination

  1. Institute cleaning procedures that match your workflow.  Knowledge of the workflow should extend beyond your lab staff to facilities staff as well. Make sure cleaning personnel know the correct order to clean floors, to clean touch points such as light switches and doorknobs regularly, and to remove trash from the lab instead of rolling large trash bins through the labs, which could spread contamination.

  2. Clean regularly with 10% bleach. It is important to wipe down any equipment or areas of suspected contamination as well as clean all work surfaces and other touch points regularly with bleach. Bleach is corrosive, so after an appropriate contact time of a few minutes, be sure to wipe those surfaces with isopropyl alcohol or water. You can monitor cleaning effectiveness by swabbing an area and testing for amplicon using PCR. Regularly make a fresh bleach solution, as it deteriorates when diluted.

  3. Clean before you work. Use a fresh bleach solution to clean work surfaces before you begin an experiment. This is particularly important in a high-traffic lab.

  4. Use good laboratory practices. Don’t wave a wet tip around under the hood. Use a paper towel or other clean absorbent barrier when putting a plate down that may be contaminated with amplicon. Limit personal items in the lab – for example, keep cell phones at your desk.

  5. Use technology to eliminate contamination. You can incorporate the use of dUTP in place of dTTP in your PCR reactions and treat each PCR with uracil-DNA glycosylase prior to amplification to eliminate any contamination from a previous PCR1.

  6. Don’t be penny wise and pound foolish. Use new tips for each transfer of solution or completed PCR sample as well as fresh gloves for each experiment. Store tips, plates, gloves, and other lab supplies away from your experimental setup to prevent contamination. In a shared lab, label your tips so that others won’t throw away your tips because they don't know where that half-used box has been.

  7. When in doubt throw it out (or bleach it). This goes for tips, plates, gloves, and even paperwork - scan and shred instead of risking contamination.

  8. Establish a written procedure. Train new lab members and provide periodic refresher training for existing members. Don’t let all of your hard work slowly slip back into “the way it’s always been done” or bad habits picked up while working in other labs.

Taking precautions to prevent amplicon contamination will minimize the need for troubleshooting and additional work.  A bit of planning will go a long way!

Reference:

1. Longo, M. C., Berninger, M. S., & Hartley, J. L. (1990). Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93(1), 125-128.

This is for information purposes only and does not constitute advice.

SMN1 and SMN2 Copy Number Determination – Simplified | 25 JUN 2018

Determining copy number variation (CNV) for the highly homologous SMN1 and SMN2 genes can pose a difficulty for those researching these targets. Because the genes only vary by a single nucleotide in their respective exon 7 regions, assay specificity is of the utmost importance. Commercial kits for determining SMN1 and SMN2 CNV by digital droplet PCR (ddPCR) or multiplex ligation-dependent probe amplification (MLPA) require specialized instrumentation, such as capillary electrophoresis systems or droplet generators and readers, to run the assays and can take in excess of a day to return results. 

We set out to simplify the protocol, reduce the turnaround time, and run these assays on instruments already in your lab. 

In November 2017, Canon BioMedical launched the Novallele™ Copy Number Assays for SMN1 and SMN2 gene targets. By using a relatively standard PCR chemistry and a high-resolution melting (HRM) analysis, we were able to create a simple workflow for these notoriously tricky targets that can be completed in less than an hour.

 

How the assays work

 

The assay is set up as a duplex reaction. Inside each assay vial, there are primer sets for the target (either SMN1 or SMN2) and a conserved, two-copy reference gene. One of the fundamental aspects of the assay is limiting the amount of dNTPs in the reaction mix. By having the target and reference amplifications compete for dNTPs,  the fluorescence values of both target and reference are used to cluster samples of the same target-gene copy number. Notice that the fluorescence values of both the target and reference genes vary in the graph to the right. It makes sense for the target’s fluorescence to change relative to the target-gene copy number, but you may be wondering why the reference gene’s fluorescence also varies. This is due to the competition for dNTPs between the target and reference gene. The black curve, which represents zero copies of SMN1, shows the maximum fluorescence at the reference peak region because all the dNTPs in the reaction mix are available for amplification of the reference gene. Inversely, the green curve has three copies of SMN1, hence more primer binding sites for the target than the reference gene, so the fluorescence is repressed for the reference amplification. 

The unique assay design provides data from two separate amplifications within each sample. This combination of data creates a fluorescent signature and tight sample clusters with the same target-gene copy number. Limiting the amplification to 25 PCR cycles is also important to how the assay works and locks in the subtle fluorescence differences between samples. If PCR was continued to the plateau phase, these important differences between the sample clusters would be lost. 

 

Data Analysis of Novallele Copy Number Assays

 

Now that we know how the CNV determination works, how are the data analyzed to make sample calls for different copy numbers? 

During plate setup, a panel of SMN1 and/or SMN2 controls are plated alongside your samples. These controls can be from a third party, such as the Coriell Institute for Medical Research, or previously characterized samples. To make a copy number call for your samples, simply determine the control with which each sample clusters.

It may be simpler to visualize this clustering using the difference plot, shown to the left. These are the same data from the derivative plot shown above, but the data is transformed using an algorithm. In this example, there are four unique clusters for 0, 1, 2, and 3 copies of SMN1. These clusters contain controls as well as samples.

 

Learn More

 

The Novallele copy number assays for SMN1 and SMN2 genes are extensively bench tested using various extraction methods and thermocyclers for accuracy and reproducibility. Therefore, along with a simplified protocol, you are assured of data quality as well. If this method is a fit for your research needs, click the links below to learn more about each product. 

 

Product

Catalog number

Number of reactions (10 uL)

 
Novallele Copy Number Mastermix 40797 200 View Details
SMN1 Novallele Copy Number Assay 40798 200 View Details
SMN2 Novallele Copy Number Assay 40799 200 View Details

 

Ready to bring these assays into your lab today? 

 

SET UP A CONSULTATION

 

For research use only.  Not for use in diagnostic procedures.
Tables, graphs, and diagrams are for illustration purposes only. 
Nothing herein constitutes medical advice.

Writing a Quality Scientific Abstract | 11 JUN 2018
As a scientist preparing for a journal article, poster presentation, or talk, you are likely required to write an abstract about the content. Make sure to check the guidelines and limitations specific to the journal, conference, or venue regarding the word limit and format, but some basic advice holds true for writing any scientific abstract. 
 
Your goal when writing an abstract is to provide enough information for a reader to know if the content is of interest and relevance to them. Give enough detail for the abstract to stand alone, like a mini article, and include the experimental hypothesis, summary of investigative methods, results summary, and conclusion. In fact, highlighting each of the sections of the paper or presentation being described is a good approach to writing the abstract. Unlike your favorite TV series, don’t leave a cliffhanger.  
 
There are publications on writing scientific abstracts and many websites that provide tips from academic and industrial institutions. They all take a step-by-step approach of essentially writing a sentence or two for each aspect. 
 
  1. Write an introductory sentence with background about why the study was performed.
  2. Write a sentence explaining the hypothesis tested.
  3. Write a sentence about the methods.
  4. Write a sentence about the results.
  5. Write a sentence about the drawn conclusions and their implications.
When complete, use built-in tools such as spell check and grammar check as your first proofreader. Proofread your own work and then ask a friend or colleague, preferably someone not as familiar with the topic as you, to read the piece. Make the edits, take a break from it, and then do a final proofread to catch the little things your brain overlooks once you get too familiar with the content. More people will read your abstract than actually read your paper or presentation, so convey the information as concisely as possible. Develop the skill of writing a solid scientific abstract to get more interest in your more in-depth content.

 

This is for information purposes only and does not constitute advice or an offer to provide services.

 
NEXTGENPCR – Discover How It Works | 4 JUN 2018
Historically, PCR instrumentation has relied on Peltier technology to power the process of heating and cooling samples, resulting in similar run times between instruments and few significant improvements in run times over the last 30 years. The NEXTGENPCR thermocycler introduces a new technology that enables samples to heat and cool almost instantly. In this blog post, we will briefly discuss the Peltier technology and discover how NEXTGENPCR eliminates ramp times.
 
The standard PCR cycle

 

standard PCR cycle

The first step of any PCR is a denaturation step that usually takes place between 94°C and 98°C. This allows the double-stranded DNA to separate into two single strands. The second step is annealing, which lowers the temperature to between 55°C and 72°C so that primers can bind to the targeted region for amplification. The final step is extension, which requires an additional temperature change. Depending on the enzyme's optimal temperature, the extension temperature can range from 68°C to 72°C. During this step, the enzyme extends the primer molecules to complete the copy of the desired fragment. These three steps are repeated for a specific number of cycles in order to amplify the target DNA fragment. 
 
A brief review of Peltier technology  
 
Today’s common thermocyclers have an aluminum or silver block with wells specifically sized for the reaction tubes or plates being used. Using a thermoelectric method, the block is heated and cooled to reach the necessary temperatures for the PCR steps, and the temperature is transferred to the contents of the tube. The time required to change the block temperature is defined as ramp time. A ramp time can take many seconds depending on the temperature change, and ramp rates vary between instruments. The fastest ramp rates on Peltier thermocyclers are on the order of 5°C or 6°C per second.  
 
In addition, some time is required to transfer the heat from the block through the tube to the sample to get the reaction to the desired temperature. Over the years, the use of thinner tubes, with a thickness of about 200 microns, has quickened the transfer of heat to the sample. The heat block must be precisely designed for the tubes being used in order to efficiently transfer the heat from the block to the sample. A drawback is the possibility of variability in the heating of the block from side to side or among the wells for each sample. A block change is also required if switching between different-sized samples as the block and sample must fit exactly.
 
How NEXTGENPCR is different
 
With NEXTGENPCR, the temperature change of the PCR is different. The samples are loaded into a microplate with wells that have a wall thickness of 40 microns, five times thinner than the usual thin-walled PCR tubes. The plate is sealed using a heat sealer and aluminum- or polyester-backed polypropylene seal that seals each well individually yet simultaneously. The microplate moves between thermal zones that are maintained at the precise temperature for each PCR step, which eliminates ramp times. The plate moves between each thermal zone in less than a second. In each thermal zone, the microplate gets pressed between two heat blocks at the correct temperature, which compresses the wells slightly and immediately brings the sample temperature to the desired temperature and mixes the sample. 

 

NEXTGENPCR technology

 

The heat blocks are flat, enabling the use of any sample plate configuration. For example, a 96-well format or 384-well format is possible without requiring any change except the sample plate. The currently available 96- and 384-well plates match standard microplate formats. The microplates have a structural, outer frame of rigid plastic and are capable of being handled by robotics. The plate face is made of thin polypropylene into which the wells are formed. The heat blocks sandwich only the inner polypropylene section of the plate so heat transfer is instantaneous.

 

NEXTGENPCR cycle

By eliminating ramp rates and optimizing the conditions for NEXTGENPCR, the typical PCR protocol can be shortened by 70-80%. A 100 bp fragment using a three-step, 30-cycle protocol was amplified in less than two minutes. By increasing speed, it is possible to add flexibility and throughput in your lab.  

 
 
 
 
Products mentioned are for Research Use Only.  Not for use in diagnostic procedures.
The NEXTGENPCR products are manufactured by Molecular Biology Systems, B.V.
Tables, graphs, and diagrams are for illustration purposes only.
All referenced product names, and other marks, are trademarks of their respective owner.
The CYP2D6 Gene's Effect on the Metabolism of Tamoxifen | 21 MAY 2018

The cytochrome P450 2D6 enzyme, encoded by the CYP2D6 gene, is responsible for metabolizing many drugs, one of which is the drug tamoxifen (1). Tamoxifen is used in the treatment of early-stage estrogen-receptor-positive breast cancer. In our previous blog titled CYP2D6 Genetic Variations and Drug Metabolism, we discussed the high variation in the CYP2D6 gene from person to person and between different ethnic groups.  In that blog, we presented how the different star-alleles are used to calculate an activity score to determine the resulting phenotype, as shown in the scale below. 

 

 

Tamoxifen is metabolized by the liver through a pathway involving the P450 2D6 enzyme into endoxifen. Endoxifen has greater antiestrogenic potency and antitumor activity than tamoxifen, making the functionality of the P450 2D6 enzyme an important factor in tamoxifen efficacy. Certain CYP2D6 genotypes can reduce the metabolic activity of P450 2D6, making the drug less effective.  By reviewing hundreds of published articles about the relationship of the CYP2D6 genotype to the metabolic conversion of tamoxifen to endoxifen, scientists made specific recommendations about the applicability of tamoxifen treatment based on an individual's genotype (2). By making these recommendations for tamoxifen treatment versus other potential options for early-stage estrogen-receptor-positive breast cancer, medicine becomes more precise. To support this research, Canon BioMedical currently offers products to continue moving this type of research forward.
 
While the CYP2D6 gene and its functionality in biology can seem complex, genotyping samples for common CYP2D6 mutations can be easily performed using an HRM-enabled thermocycler. The currently available Novallele genotyping assays for CYP2D6 targets, as well as homozygous-mutant and wild-type characterized controls, are listed in the table below. If there are additional CYP2D6 targets that you would like to investigate as part of your pharmacogenetic research, email us to find out if we can help. 

  

 Star allele  dbSNP number  HGVS (c.)  GenBank number  Activity  Assay  Control set
 *2  rs16947  c.886C>T  2850C>T  Normal  40054  40422
 *2  rs1135840  c.1457G>C  4180G>C  Normal  40390  40730
 *2A  rs1080985  c.-1584C>G  -1584C>G  Normal  40237  40595
 *4  rs3892097  c.506-1G>A  1846G>A  Nonfunctional  40403  40743
 *6  rs5030655  c.454delT  1707delT  Nonfunctional  40129  40491
 *7  rs5030867  c.971A>C  2935A>C  Nonfunctional  40061  40429
 *8  rs5030865  c.505G>T  1758G>T  Nonfunctional  40378  40719
 *9  rs5030656  c.841_843delAAG  2615-2617delAAG  Reduced  40365  40707
 *10  rs1065852  c.100C>T  100C>T  Reduced  40327  40678
 *11  rs5030863  c.883G>C  883G>C  Nonfunctional  40405  40745
 *12   rs5030862  c.124G>A  124G>A  Nonfunctional  40796  40811
 *17  rs28371706  c.320C>T  1023C>T  Reduced  40156  40517
 *29  rs59421388  c.1012G>A  3183G>A  Reduced  40188  40546
 *41  rs28371725  c.985+39G>A  2988G>A  Reduced  40315  40669
 *52  rs28371733  c.3877G>A  3877G>A  Reduced  40404  40744
 
 

To learn more, register to download our infographic about pharmacogenetics.  

 

References:

 

  1. Owen, R. P., Sangkuhl, K., Klein, T. E., & Altman, R. B. (2009). Cytochrome P450 2D6. Pharmacogenetics and genomics, 19(7), 559.
  2. Goetz, M.P., et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for CYP2D6 and Tamoxifen Therapy.
 
Products mentioned are for Research Use Only.  Not for use in diagnostic procedures.
Tables, graphs, and diagrams are for illustration purposes only. 
Nothing herein constitutes medical advice.

 

 

 
Creating a HET from Novallele™ WT and HOM Controls | 14 MAY 2018
Canon BioMedical offers Novallele Control DNA kits for all of our genotyping assays. These kits contain a homozygous wild type (WT) and homozygous mutant (HOM) control for the assay of interest. While having WT and HOM controls should be sufficient references to make genotyping calls, these controls can be mixed together to create a heterozygous mutant (HET) control, which can be useful for comparison to test samples. The Novallele control kit includes 120 µL of homozygous wild-type (WT) and homozygous variant (HOM) synthetic control DNA, sufficient template DNA for 30 control reactions. 
 
The first step to preparing a HET control is to mix equivalent volumes of WT and HOM control DNA in a new tube. A standard 10 µL reaction requires 3.3 µL of DNA template, therefore, to prepare enough control DNA for a triplicate set of reactions combine 6 µL of WT and 6 µL of HOM DNA for a total of 12 µL. Pipette 3.3 µL into each reaction tube (assumes all reactions are performed in triplicate). An example of this is shown in Figure 1 using the control set (catalog number 40734) with the APOE c.388T>C assay (catalog number 40394). In the majority of cases, the 1:1 ratio of WT:HOM yields optimal HET control DNA. 

 

Derivative melt curve plot of the ApoE c.388T>C assay

 
Figure 1. Derivative melt curve plot of  the APOE c.388T>C assay. 10 µL reactions were prepared using 3.3 µL each of WT (black), HOM (red), or the prepared HET (blue) control DNA template  and run on a BioRad CFX thermocycler following the conditions recommended in the assay-specific protocol. The HET was generated from a 1:1 ratio of WT:HOM control DNA.
 
There are rare cases when the 1:1 ratio of WT:HOM control DNA is not the optimal ratio to generate a balanced HET control DNA sample (Figure 2).  Figure 2 clearly shows that the HET is unbalanced. The WT peak is much higher intensity than the HOM peak making the HET sample less distinguishable from the WT genotype and not representative of an actual HET.
 

Derivative melt plot of the DPYD c. 1905+1G>A assay

 
Figure 2. Derivative melt plot of the DPYD c. 1905+1G>A assay (catalog number 40180). 10 µL reactions were prepared using 3.3 µL each of WT (black), HOM (red), or HET (blue) control DNA template  and run on a BioRad CFX thermocycler following the conditions recommended in the assay-specific protocol. The HET was generated from a 1:1 ratio of WT:HOM control DNA (catalog number 40538).
 
To optimize the HET control from unbalanced to balanced, it is recommended to run a few different ratios of HIGH:LOW peak (Table 1). Although it seems logical to use the delta Ct to calculate the appropriate ratio of WT:HOM, empirically this significantly overshoots the target and leads to a HET that is unbalanced in the other direction. Thus, it is recommended to test a standard set of ratios to determine the optimal ratio to generate a balanced HET control.  For the DPYD example, ratios of HIGH (WT):LOW (HOM) of 1:1, 1:1.2, 1:1.4 were tested and the appropriately balanced ratio was 1:1.4 (Figure 3). 
 
 

Ratio HIGH:LOW

 

µL Stock HIGH (WT*)

 

µL Stock LOW (HOM*)

 

1:1 6** 6**
1:1.2 4.8 7.2
1:1.4 3.6 8.4
Table 1. The ratios and volumes of stock of WT:HOM to optimize a HET control are shown.  *The DPYD example shown. ** Assumes all reactions will be performed in triplicate.

 

HET control titration

Figure 3. Derivative melt plot of the DPYD c. 1905+1G>A assay (catalog number 40180). 10 µL reactions were prepared using ratios of WT: HOM control DNA template (catalog number  40538) of 1:1 (blue), 1:1.2 (purple), 1:1.4 (pink) and run on a BioRad CFX thermocycler following the conditions recommended in the assay-specific protocol. The balanced HET (pink) was generated from a 1:1.4 ratio of WT:HOM control DNA.
 
In summary, in many cases, Novallele control set users can create a HET sample by mixing WT and HOM controls at a 1:1 ratio. For the rare instances in which the 1:1 ratio of WT:HOM does not provide a balanced HET control, it is simple to optimize the ratio and generate a satisfactory HET control. The Novallele control kits enable labs to easily start genotyping studies by eliminating the need to source characterized controls from biorepositories or colleagues.
 
For any questions about this process, please contact our application specialists at contactus@canon-biomedical.com or call
844-CANONBIO.      
 
For additional technical resources such as research area specific product lists and applications data, click the link below to visit our resources page.
 

Learn More

 

 

 

Unique Targets for Ashkenazi Jewish Population Gene Mutation Studies | 7 MAY 2018
The Ashkenazi Jewish population is one of the most intensely studied ethnic groups from a medical genetics standpoint. This group of individuals originates from an area near the Rhine River between northern France and the western part of Germany. Diseases such as Bloom syndrome, cystic fibrosis, Canavan disease, and Tay-Sachs disease have a higher incidence rate within the Ashkenazi Jewish population than in the general population. From a carrier perspective, one in 30 of those of Ashkenazi descent are carriers of Tay-Sachs disease, whereas the general population has a carrier rate of one in 300.  
 
Mutations common to the Ashkenazi Jewish population are known to vary dramatically, and the severity of the associated disease is often dependent on the actual gene mutation. There are several different methods to detect mutations associated with genetic disorders common to the Ashkenazi Jewish population.  These methods range from complicated full-genome technologies, like next-generation sequencing, to tailored approaches, like real-time, hydrolysis probe-based PCR and high-resolution melting (HRM) analysis.
 
The Novallele genotyping assays are HRM based and include more than 60 assays developed to detect mutations common to the Ashkenazi Jewish population. Twelve of these assays do not have a real-time, hydrolysis probe-based PCR genotyping equivalent available.
 
Unique Novallele Genotyping Assays:
 
 Assay name  RefSNP ID number  Assay catalog number  Control set catalog number
 FANCC c.456+4A>T Novallele Genotyping Assay  rs104886456  40147  40508
 FANCC c.67delG Novallele Genotyping Assay  rs104886459  40091  40455
 GBA c.115+1G>A c.115+1G>T Novallele Genotyping Assay  rs104886460  40101  40464 & 40750
 GBA c.1226A>G Novallele Genotyping Assay  rs76763715  40324  40675
 GBA c.1263-1319del55 Novallele Genotyping Assay  rs80356768  40384  40724
 GBA c.1448T>C c.1448T>G Novallele Genotyping Assay  rs421016  40301  40657 & 40769
 GBA c.1604G>A Novallele Genotyping Assay  rs80356773  40368  40710
 GBA c.84dupG Novallele Genotyping Assay  rs387906315  40102  40465
 HEXA c.1274_1277dupTATC Novallele Genotyping Assay  rs387906309  40184  40542
 HEXA 7.6kb del Novallele Genotyping Assay  No rs ID  40320  40672
 IKBKAP c.2204+6T>C Novallele Genotyping Assay  rs111033171  40181  40539
 SMPD1 c.996delC c.995 C>G Novallele Genotyping Assay  rs387906289  40344  40689 & 40774
 
Click on the Assay catalog number link to learn more about each assay.
 
Need to detect genetic variants common in the Ashkenazi Jewish population as part of your research?  Learn more about our simple and accurate method by clicking the button below to see a full list of related assays. 
 
 


Products mentioned are for Research Use Only. Not for use in diagnostic procedures.
Nothing herein constitutes medical or legal advice.

Meet the Inventor of NEXTGENPCR [Interview Part 2 of 2] | 30 APR 2018
In last week's blog, we began a very interesting interview with Gert de Vos, the inventor of NEXTGENPCR and Director at Molecular Biology Systems, B.V. (MBS). This week Dana Pfister Sullivan, Product Manager at Canon BioMedical, finishes our interview with Gert about the ultrafast NEXTGENPCR.
NGPCR instrument
 

DS: NEXTGENPCR allows PCR to go much faster than what researchers are used to. In your opinion, why does faster PCR matter?

We have been speaking to a lot of customers over the past few years. From what I hear, there are two things that are important to them when it comes to their work. One has to do with speed, and the other has to do with throughput; these are two sides of the same coin. If you can do PCR very fast using one instrument, that same instrument can do a lot more PCRs per day. So, for example, if you look at crop developers, they want to do a lot of PCR runs every day. If one instrument can do the work of  ten or, sometimes even, twenty instruments, then you can save a lot of space and energy. 
 
That is one side of the coin. The other is simply that some users would really like to go faster. We have a current user in the north of the Netherlands that will publish their data in the next couple of months. They are running their assay using our instrument for very quick detection of E. coli in urinary tract infections – and it is so fast that they can detect E. coli in less than 30 minutes. It doesn't take a lot of imagination to see how this could be duplicated to other infectious disease research - like lung infection or sepsis research for example.
 
Another consideration when it comes to speed and run times specifically is instrument availability. Most lab’s cyclers are being reserved for part of the day, usually half the day, for PCR experiments. This is because PCR experiments can take close to three hours. If one could reduce that time to ten or twenty minutes, then you would not need such complicated systems – you would only need a few cyclers. And if someone wants to use it, they would simply go. And if it’s not free, you can wait because it will be free shortly. 
 
DS: How have you seen NEXTGENPCR impact a current customer’s research? What is the response you are receiving from current users?
 
It is absolutely fantastic. We have one user doing detection of Clostridium difficile using downstream ribotyping fragment analysis. Currently, this is a two-day process. Using NEXTGENPCR, they are able to squeeze the whole two days into one day. Of course, they still do the fragment analysis, which takes time after that; you need close to four to six hours. But if you can do your PCR so much faster, then that’s not a problem. 
 
DS: How do you see  NEXTGENPCR products making an impact on PCR-based research?
 
Look at developments that take an established idea and reinvent what that technology does – look at 3D printers. 3D printers are so nice because we all like to build things. 3D printers let your imagination come to life and see things at all angles. NEXTGENPCR may be comparable to that. Currently, if you do PCR in your research and plan a lot of PCR reactions, meaning you spend two to three days per week running PCRs, you have to wait for the results to determine how to continue. With NEXTGENPCR, you can do a PCR in ten minutes, run a gel, and immediately start thinking about what to do next — the whole thinking around your project changes. NEXTGENPCR will affect the way people think a lot  –  because the results are available earlier, your project will shrink in time, and NEXTGENPCR will give you the opportunity to do other things within your research using this saved time.
 
DS: NEXTGENPCR is an end-point PCR instrument. What downstream methods have you tested after PCR using the instrument?
 
We all know all the detection methods; for example, I mentioned ribotyping fragment analysis. Sanger sequencing is another application requiring a PCR step. We have performed a post-PCR high-resolution melting analysis. Sometimes even a simple agarose gel is enough to see your result. If you need a yes or no answer, you could decide to seal the plate with transparent film, mix some dye in, and put the plate on a blue reader. There are a mix of different analysis methods that are now being used with typical end-point PCR instruments, and these methods do not change when using a NEXTGENPCR instrument. 
 
DS: Thank you so much for chatting with us. Canon BioMedical is very happy to be the exclusive US and Canadian distributor of this exciting new technology.
 

It's time to go faster

 
 
 
Products mentioned are for Research Use Only.  Not for use in diagnostic procedures.
The NEXTGENPCR products are manufactured by Molecular Biology Systems, B.V.
All referenced product names, and other marks, are trademarks of their respective owner.
 
Meet the Inventor of NEXTGENPCR [Interview Part 1 of 2] | 23 APR 2018
Recently, Dana Pfister Sullivan, a product manager at Canon BioMedical, sat down with Gert de Vos, the inventor of the NEXTGENPCR instrument and Director at Molecular Biology Systems, B.V. (MBS). Gert has master’s degrees in biology and physics from Leiden University in the Netherlands. After teaching physics in Curaçao, Gert returned to the Netherlands and embarked on a career as an entrepreneur and inventor in the life sciences. 
 
DS: How did you come up with the idea for NEXTGENPCR?
 
Well, I was in a meeting focused on the detection of pathogens in milk. During the meeting, we were discussing gel electrophoresis and touched briefly on PCR. One person at the table mentioned PCR chips. I was aware of how PCR chips worked, specifically how the liquid or sample is pumped through different temperature zones, and also that they required nanoliter volumes rather than microliter volumes. I thought, wouldn’t it be great if we could do something similar — not only move the liquid around, but instead move the whole enclosure. That is when I got the idea — so then I went home, and we started on prototypes for what would become NEXTGENPCR. The first experiment we did was around ten minutes, and it worked brilliantly. I said, “Wow, we have something here!” 
 
DS: How is NEXTGENPCR different than standard end-point PCR instruments?
 
In a typical PCR instrument there is an aluminum or silver block with cavities that holds the individual tubes of the PCR plate. These tubes are made of polypropylene, and they have a 200 to 300 micron wall to help hold their shape. The block is heated to the desired temperature, usually 95°C for denaturation, and then cooled down to the desired annealing temperature. What happens then is interesting; the cooling is done by Peltier elements. The moment you start to use a Peltier element for cooling, the other side gets hot; so, you have to cool a lot more than just the block. When the other side gets hot, the efficiency tumbles; so, while it is initially cooling at eight degrees per second, after a few seconds it slows down to two to three degrees per second. Therefore, it takes some time for the instrument to achieve the correct annealing temperature. Also, the sample inside the tube relies on convection to reach the correct temperature. Rather than use the common Peltier technology, NEXTGENPCR uses temperature zones that eliminate the time it takes to heat and cool during a three-step PCR.  
 
DS:  How does NEXTGENPCR achieve such fast PCR?
 
The technology differs from typical thermocyclers in two ways that enable fast PCR. One, the instrument has three temperature zones, one each for denaturing, annealing, and extension. Two, we decrease the thickness of the well so that the liquid can reach the needed temperature quicker. Our plates have polypropylene enclosures where the PCR mix has been added inside. These enclosures are squeezed between two blocks at the correct temperature. So, if the sample has to go from 95°C to 60°C, the plate moves from the 95°C temperature zone to the 60°C temperature zone. The instrument then presses the blocks together, slightly deforming the enclosures, which allows the PCR reagents to not only mix instantly but also come to the desired temperature, essentially removing the ramp time.
 
DS: What was important to you when you were designing NEXTGENPCR? 
 
That is an excellent question. While developing the NEXTGENPCR instrument, I met an expert in the PCR market. I think he sold his first PCR instrument in 1984 or 1985. He told me this is potentially a market-disruptive technology, but not in its current shape. He noted the need to have a microplate format because then it will be compatible with all the current downstream applications. We started to work together because of what he said. We started making an instrument that accepts microplate formats. Our plate matches the rim of your typical microplate; in the middle there is a polypropylene sheet with either 96 or 384 wells. Our blocks are flat faced, so it does not matter what plate format you use in the instrument; there are no changes needed. 
 
In addition to the plate, it was important to achieve high-speed PCR while also considering those things important to a user such as how much space the instrument takes on the bench, how much energy is used, and, in the end, how much the device will cost. 
 
DS: How are the plates and consumables used with the NEXTGENPCR instrument different than standard 96- and 384-well plates?
 
The plates are the same size as a typical microplate because we use the same outside rim. They adhere to the standard defined by the Society for Laboratory Automation and Screening, so they fit in all robotics – both upstream and downstream. However, they differ in the center where we have inserted polypropylene film that has either 96 or 384 wells with a well thickness of 30 to 40 microns. The user’s current PCR mix is pipetted into the wells and then sealed with a heat sealer to close the wells. 
 
DS: You mentioned that energy usage was an important factor in the design. How is the instrument able to save energy?
 
That is simple to explain — since the instrument has three temperature zones, we don’t need to change the temperature of the blocks. This means we isolate the blocks in the denaturing zone and the extension zone, and in the annealing zone we are able to cool at a certain rate so that the instrument is capable of touchdown and stepdown experiments. Because of this isolation, the instrument is running at 150 to 170 watts rather than 800 to 1200 watts that a standard cycler uses. Then, if you take into account that a PCR experiment lasts between five and 20 times shorter with NEXTGENPCR, you can imagine the amount of energy you use. It could be up to 100 times less compared to other PCR instruments.
 

-------------------

This is only the first part of our interview with Gert. Come back next week for the conclusion of our interview. In the meantime, download the application note that explains how a 100 bp fragment was amplified in less than 2 minutes using NEXTGENPCR.
 

 

DOWNLOAD APPLICATION NOTE

 

 
Products mentioned are for Research Use Only. Not for use in diagnostic procedures.
The NEXTGENPCR products are manufactured by Molecular Biology Systems, B.V.
All referenced product names, and other marks, are trademarks of their respective owner.

 

 

 

CYP2D6 Genetic Variations and Drug Metabolism | 16 APR 2018
The CYP2D6 gene encodes for the cytochrome P450 2D6 enzyme, which is responsible for metabolizing as many as 25% of all prescribed drugs.1Variation in the CYP2D6 gene is notoriously high from person to person and between different ethnic groups. The high degree of genetic variability coupled with the number of medications the enzyme metabolizes makes the gene one of the most important pharmacogenetic targets. 
 
The therapeutic effect of drugs that are transformed into their active form by the cytochrome P450 2D6 enzyme, such as codeine, can be drastically altered due to variations of the CYP2D6 gene. As an example, an individual with the ultrarapid metabolizer phenotype that is prescribed codeine would likely create too much of the active metabolite, morphine, and suffer toxic side effects. Inversely, someone with a poor or intermediate metabolizer phenotype may not receive the complete pain relief expected due to less morphine being biologically available. 
 
Metabolic phenotypes as a result of CYP2D6 variation fall into four categories: poor, intermediate, normal, and ultrarapid metabolizers. To assign a metabolic phenotype, pharmacogenetic researchers must run a genotyping panel as well as a copy number analysis of the CYP2D6 gene in order to generate an activity score. The first step in assigning an activity score requires knowing which mutations, designated as star alleles or "*alleles", exist in the sample. In addition,  deletions or duplications of the gene must be considered. There are over 100 different star alleles for the CYP2D6 gene, and some of the more common star alleles and their respective activity levels are shown below.
 

 

 Activity level  Activity score per allele  Common *alleles
 Normal  1  *1, *2
 Reduced  0.5  *9, *10, *17, *29, *41
 Nonfunctional  0  *3, *4, *5, *6, *7, *8, *14, *36


Poor, intermediate, normal, and ultrarapid metabolizer phenotypes are defined by the activity score values of each of the alleles present in the sample added together, and, if duplication or deletion of alleles exist, they must be taken into consideration. Examples of genotypes and how the associated activity score is calculated and used for the assignment to a metabolizer phenotype can be seen in the scale below. Note in the ultrarapid metabolizer example that the copy number of 2 for one allele adds its score twice.



 

 

While the CYP2D6 gene and its functionality in biology can seem complex, genotyping samples for the common mutations can be done in any lab with an HRM-enabled thermocycler. The Novallele genotyping assays for CYP2D6 targets currently available, as well as homozygous mutant and wild-type characterized controls are listed in the table below. If there are additional CYP2D6 targets that you would like to investigate in your pharmacogenetic research, let us know by emailing us.   

 

 

 
 Star allele  dbSNP number  HGVS (c.)  GenBank number   Activity  Assay  Control set
 *2  rs16947  c.886C>T  2850C>T  Normal  40054  40422
 *2  rs1135840  c.1457G>C  4180G>C  Normal  40390  40730
 *2A  rs1080985  c.-1584C>G  -1584C>G  Normal  40237  40595
 *4  rs3892097  c.506-1G>A  1846G>A  Nonfunctional  40403  40743
 *6  rs5030655  c.454delT  1707delT  Nonfunctional  40129  40491
 *7  rs5030867  c.971A>C  2935A>C  Nonfunctional  40061  40429
 *8  rs5030865  c.505G>T  1758G>T  Nonfunctional  40378  40719
 *9  rs5030656  c.841_843delAAG  2615-2617delAAG  Reduced  40365  40707
 *10  rs1065852  c.100C>T  100C>T  Reduced  40327  40678
 *11  rs5030863  c.883G>C  883G>C  Nonfunctional  40405  40745
 *12  rs5030862  c.124G>A  124G>A  Nonfunctional  40796  40811
 *17  rs28371706  c.320C>T  1023C>T  Reduced  40156  40517
 *29  rs59421388  c.1012G>A  3183G>A  Reduced  40188  40546
 *41  rs28371725  c.985+39G>A  2988G>A  Reduced  40315  40669
 *52  rs28371733  c.3877G>A  3877G>A  Reduced  40404  40744
 

To learn more, register to download our infographic about pharmacogenetics.  

References:
1. Owen, R. P., Sangkuhl, K., Klein, T. E., & Altman, R. B. (2009). Cytochrome P450 2D6. Pharmacogenetics and genomics, 19(7), 559.

For research use only.  Not for use in diagnostic procedures.
Tables, graphs, and diagrams are for illustration purposes only. 
Nothing herein constitutes medical advice.

The APOE gene — Mutations Lead to Increased Risk of Alzheimer’s Disease and Recurrent Stroke                  | 9 APR 2018

By definition, the APOE gene is responsible for the synthesis of the protein apolipoprotein E, a member of the lipoprotein family. This family of proteins is responsible for metabolism, movement, and clearance of fat-soluble vitamins and cholesterols across multiple body systems. However, when speaking about the APOE gene and its well-characterized mutations, the conversation usually focuses around the gene's role in neurological diseases, especially Alzheimer’s disease. In large cohort studies, mutations in the APOE gene, specifically those that relate to the E4 allele, have been shown to increase the risk of developing Alzheimer’s disease1

Table 1. The table shows the genotype designations with the associated mutation and the Alzheimer’s disease risk correlation and allele frequency as determined by the cohort study.1

 Genotype  c.388T>C 
 (rs429358)
 c.526C>T 
 (rs7412)
 Disease risk   Allele frequency
 E2/E2  T:T  T:T  40% less likely  8.40%
 E2/E3  T:T  T:C  40% less likely  
 E2/E4  C:T  C:T  2.6 times more likely  
 E3/E3  T:T  C:C  Average risk  77.90%
 E3/E4  T:C  C:C  2-3 times more likely  
 E4/E4  C:C  C:C  12 times more likely  13.7%, 40% in AD population

 

While the concordance of allele frequency to the risk of disease has been thoroughly researched and reproduced, there are exceptions to this rule. Specific populations, such as Nigerians, have one of the highest E4 allele frequencies yet Alzheimer’s disease remains rare in this group.

 
While the risk of developing Alzheimer’s disease based on one’s APOE allele type is well characterized, recent research has shown that these mutations play a role well beyond disease prediction. A 2017 publication from the Munoz lab shows that females with Alzheimer’s disease that also carry homozygous E4 alleles have a higher likelihood of developing a more severe disease phenotype2. Females with this high-risk genotype were more likely to experience delusions and/or hallucinations in addition to the more common characteristics of Alzheimer’s disease. To learn more about this phenomenon, visit the ClinicalOMICS webinar page and register to watch a webinar sponsored by Canon BioMedical featuring Dr. David Munoz from the University of Toronto. 
 
Going beyond Alzheimer’s disease, the APOE  E4 genotype has also been shown to potentially affect the likelihood of recurrence in individuals who have suffered a stroke. A research study at the University of Edinburgh reported in The Lancet Neurology shows that using the APOE genotype in addition to more common prognostic tools, such as CT scans, could give physicians more confidence in segmenting out which individuals have a higher risk of having another stroke3. This study was quite limited in the number of participants, and the authors mention their desire to expand these investigations across multiple external sites in the future.   
 
If, after reading up on the APOE gene and its role in neurogenetics, your lab wants to conduct research on the APOE gene, we have products to help. Genotyping samples for the common APOE mutations can be done in any lab with an HRM-enabled thermocycler. The Novallele™ genotyping assays for APOE targets currently offered, as well as characterized controls, are listed in the table below.
 
 Assay name  Protein change  Assay catalog number  Control set catalog number
 APOE c.388T>C Novallele Genotyping Assay  Cys130Arg  40394  40734
 APOE c.526C>T Novallele Genotyping Assay  Arg176Cys  40395  40735
 
Novallele genotyping assays and reagents are For Research Use Only. Not for use in diagnostic procedures. 
 
References:
 
1. Liu, C. C., Kanekiyo, T., Xu, H., & Bu, G. (2013). Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nature Reviews Neurology, 9(2), 106. 
2. Kim, J., E Fischer, C., A Schweizer, T., & G Munoz, D. (2017). Gender and Pathology-Specific Effect of Apolipoprotein E Genotype on Psychosis in Alzheimer’s Disease. Current Alzheimer Research, 14(8), 834-840. 
3. Rodrigues, M. A., Samarasekera, N., Lerpiniere, C., Humphreys, C., McCarron, M. O., White, P. M., & Smith, C. (2018). The Edinburgh CT and genetic diagnostic criteria for lobar intracerebral haemorrhage associated with cerebral amyloid angiopathy: model development and diagnostic test accuracy study. The Lancet Neurology.

 

Nothing herein constitutes medical advice.

 

 

A Guide to the Novallele™ HRM Analyzer | 26 MAR 2018
A helpful high-resolution (HRM) analysis software package should be easy to use, make the output simple to understand, and provide publication-quality results. The Novallele HRM Analyzer software offers an intuitive and efficient alternative to the often cryptic data analysis software that comes standard with many thermocyclers. 
 
The Novallele HRM Analyzer is free, cloud-based software that offers genotyping and copy number analysis through an intuitive user interface. Step one is to upload data as a text file by either dragging and dropping a file or using standard file uploading procedures. The software currently accepts text files that are unedited and exported from the Bio-Rad CFX, QuantStudio™, LightCycler® 480 System, or even the ring-based Rotor-Gene® Q. Sample names transfer with the data so hovering over the well positions with your mouse allows easy selection of no-template control (NTC) and allele control wells for review. Samples are selected or deselected by clicking, and entire rows can be added or removed as required for analysis. The commonly used normalization bars, T1,T2,T3, and T4, are shown on the raw melt window. The four-panel view lets you monitor the normalization on both the normalized melt curve window and the derivative melt curve window (Figure 1). If you struggle properly placing the normalization bars, using the derivative curves is an immense help. 
 

Figure 1. Sample analysis of QuantStudio 7 genotyping data of one row of samples minus NTCs as shown in the "Plate Selection" panel. The upper-left graph shows raw melt data with normalization bars. The upper-right graph shows normalized melt data. The lower-left graph shows derivative curves, and the lower-right graph shows the difference curves without the cluster analysis. 

The truly novel feature of the Novallele HRM Analyzer is the unique functionality of its clustering feature (Figure 2). Although other thermocycler software packages offer this feature, clustering is usually cumbersome to apply and rarely allows you to modify the cluster number or  label the unknown samples. In contrast, the Novallele HRM Analyzer clustering tool provides up and down arrows to change the cluster numbers, a simple checkbox to set the baseline value, and a dropdown list of labels for control samples as well as the unknowns that only require a click to assign a genotype or copy number. The graphs are easily saved with a right click and an especially nice feature is that the statistics behind the data can be downloaded to an excel sheet. Both the numerical values for the derivative curves and clusters are available for download; thus, all analyzed data can be saved in two different formats: graphical and numeric. 

Figure 2. SMN1 copy number data from the RotorGene Q that was analyzed using the clustering feature. An example of downloadable statistics is shown in the upper-left table, hiding the raw melt data. The normalized melt curves and derivative curves are shown with the cluster analysis turned on as compared to Figure 1. The ring layout of the samples is in the upper-right table with the cluster identification in the table below.

In summary, the Novallele HRM Analyzer offers an easy-to-use, complete analysis method for both genotyping and copy number variation. This simple software includes all the common features found in thermocycler programs plus the extra benefits of numerical data tracking and adjustable clustering baselines. The Novallele HRM Analyzer doesn’t require any heroic data formatting, as a text file is sufficient, and the import works for four qPCR instruments. More detailed instructions and examples can be found in the online guide. Check out the Novallele HRM Analyzer for free today!

All referenced product names and other marks, are trademarks of their respective owners.
Nothing herein constitutes medical advice.

Genotyping Your Way to Better Medicine [Free Infographic] | 19 MAR 2018
With a shift toward precision medicine, physicians are no longer using a "one size fits all model" to treat patients. People do not respond to drugs in a uniform and predictable manner, and single nucleotide polymorphisms (SNPs) or other genomic insertions or deletions can alter a person’s metabolic pathways that function in the breakdown of a drug. 
 
Pharmacogenetics is the study of drug response variability in relation to specific genes. By combining pharmacology and genetics, pharmacogenetic researchers work to develop and understand safe, effective medications and dosages that are tailored according to an individual’s DNA makeup.
 
Register to download our infographic about pharmacogenetics, drug metabolism, and examples of gene influences on particular drugs.


Tables, graphs, and diagrams are for illustration purposes only. 
Nothing herein constitutes medical advice.

Updates in Newborn Screening of SMA across the U.S.A. | 12 MAR 2018
Across the U.S.A., state-sponsored pilot studies are underway to lay the groundwork for adding spinal muscular atrophy (SMA) to the nationwide Recommended Uniform Screening Panel (RUSP). In preparation for the possibility that SMA is added to the RUSP, states like New York, Utah, Massachusetts, and North Carolina have conducted or will begin studies to see how labs would integrate SMA testing into their newborn screening workflows.    
 
Taking screening efforts into their own hands, Missouri passed Senate Bill 50 in July 2017 that provides all newborns in the state will get screened for SMA along with more common, routinely tested diseases such as beta thalassemia, cystic fibrosis, and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In late December 2017, Minnesota followed suit and became the second state to offer statewide newborn screening of SMA. Soon thereafter in late January 2018, Utah announced that every newborn in the state would be tested for SMA.
 
In addition to state-sponsored studies, industry organizations and advocacy groups representing various facets of SMA healthcare have also begun to ratchet up pressure to begin nationwide SMA screenings. In February 2017, the American College of Obstetricians and Gynecologists (ACOG) released their guidance recommending carrier screening for all women expand to include SMA-related deletions. In February 2017, Cure SMA launched the SMA Newborn Screening Coalition that will advocate at both the state and federal level in an effort to put SMA screening on the RUSP.
 
In late-breaking news, the Advisory Committee on Heritable Disorders in Newborns and Children testified on February 9, 2018 to the Department of Health and Human Services that they recommended adding SMA to the RUSP. 
 
One reason that things are moving so quickly to get states and the nation at large on board for newborn screening of SMA is the recently approved drug Spinraza®. When used on children diagnosed with SMA at a point when they are still pre-symptomatic, the drug has demonstrated, in some cases, that these children can achieve age-appropriate motor function milestones. Newborn screening for SMA will be integral in getting pre-symptomatic SMA-affected children on this drug within days of birth instead of waiting until symptoms arise months or even years later.
 
To learn more about therapy advances in treating SMA-affected individuals, check out our recent blog titled Developments in Spinal Muscular Atrophy (SMA) Therapeutics.

 

Nothing herein constitutes medical or legal advice.

 

6 Key Benefits of High-Resolution Melting | 5 MAR 2018

Since sequencing the human genome, scientists have identified specific mutations of particular genes that affect human health. Through in-depth investigation of genetic variants, scientists continue to identify and understand disease states and develop new drugs, such as targeted or personalized therapeutics. There are a variety of genetic variant detection methods such as complicated full-genome technologies, like next-generation sequencing, and tailored approaches, like hydrolysis probe PCR and high-resolution melting (HRM).

HRM is a simplified detection method that can provide a fast answer for known genetic mutations, such as single-nucleotide polymorphisms (SNPs) and indels. HRM detects fluorescence changes based on target melting - the exact fluorescence change varies depending on the DNA sequence and assay used. When compared to the reference DNA, a shift in melting temperature or a change in the melt curve shape indicates a genetic variation.

 

 

Here are six key benefits of HRM:

 

 

bullseye

Accurate

HRM is highly accurate. In one study that looked at 35 publications, HRM was found to be a highly sensitive technology able to detect disease-associated mutations in humans. Overall, the summary sensitivity was 97.5%. HRM specificity showed considerable heterogeneity between studies.

 

dollar sign

Cost effective

HRM is cheaper per sample when compared to other genotyping methods, such as Sanger sequencing, which makes HRM ideal for large-scale genotyping projects.

 

 

timer

Fast

Requiring just an hour from sample to answer, HRM is quick because there is no need to process or separate PCR products. Once PCR is finished, an HRM step is performed, and analysis is completed using the free, web-based Novallele HRM Analyzer.

 

 

Minimal reagents

HRM generates little waste and only requires the reagents needed for a PCR reaction thus eliminating the need for HPLC solvents or denaturing gradient gel electrophoresis.

 

 

PCR Machine

Simple

No special instrumentation is required for HRM. HRM analysis can be done on any thermocycler with real-time and fluorescent capture capabilities. A high-resolution melt step can be added to the end of a PCR run and analyzed immediately after completion.

 

 

Flexibility

Versatile

HRM can be used to detect SNPs and indels as well as methylation analysis and mutation scanning for copy number analysis.

 

Reference: Li B-S, Wang X-Y, Ma F-L, Jiang B, Song X-X, et al. (2011) Is High Resolution Melting Analysis (HRMA) Accurate for Detection of Human Disease Associated Mutations? A Meta Analysis. PLoS ONE 6(12): e28078.

 

 

Female-Lab-Tech-Viewing-Computer-Monitor

Want to learn more? Register to watch a
free HRM analysis webinar.

 

Developments in Spinal Muscular Atrophy (SMA) Therapeutics | 2 MAR 2018

 

In a our last blog, Genetic Basis of Spinal Muscular Atrophy (SMA), we described the lack of functional survival motor neuron (SMN) protein as the cause of SMA. Full-length transcripts that yield functional SMN protein are primarily synthesized by the SMN1 gene. Affected individuals have zero copies of the SMN1 gene and, therefore, lack enough functional SMN protein to protect their motor neurons from undergoing atrophy. The SMN2 gene acts as a backup but only produces a small amount of functional protein due to a splicing variation that limits production of the full-length transcript. Those affected by SMA but with high copy numbers of SMN2 can produce enough functional SMN protein to delay the onset of the disease or lessen disease severity. 

Numerous therapeutics in various states of development can be grouped into two main categories:

 

  • Therapies that address the genetic basis of the disease  These therapies attempt to either correct a mutated or replace an absent SMN1 gene or increase the amount of functional protein derived from the SMN2 gene.       
  • Therapies that protect the physical systems affected by the lack of SMN protein  These therapies work to either shield motor neurons normally affected by the lack of functional SMN proteins or prevent or restore muscle loss in SMA-affected systems.

The diagram below from the Cure SMA organization outlines current SMA drugs in development.

SMA drugs in development

 

You’ll notice that one therapeutic, Spinraza® (nusinersen), developed by Biogen/Ionis gained FDA approval in December of 2016. Spinraza works by correcting the splicing error associated with the SMN2 gene that reduces the amount of full-length SMN transcript. The drug accomplishes this by using an antisense oligonucleotide that corrects the alternate splicing present in SMN2 RNA. By correcting the splicing, the drug effectively converts the altered SMN2 mRNA to SMN1 mRNA, thus increasing the amount of full-length transcript available to create the functional SMN protein.

Next in the SMA blog series, we’ll look at the changing landscape of newborn screening for SMA. 

Nothing herein constitutes medical advice.

Image used with permission from Cure SMA (curesma.org).

Genetic Basis of Spinal Muscular Atrophy (SMA) | 1 MAR 2018

 

Individuals with spinal muscular atrophy (SMA) suffer from a nerve-wasting disorder that ultimately makes their skeletal muscles atrophy, leading to impaired movement and possibly death. The nerves affected in this disease are neuronal cells in proximity to the spinal cord. These nerves undergo degradation due to a lack of the survival motor neuron (SMN) protein.

The disease typically only affects individuals that have a homozygous deletion of the SMN1 gene (approximately 95% of cases); however, other cases of SMA can arise from having mutations within the SMN1 gene that effectively make the gene nonfunctional. SMA is an autosomal recessive inherited disease, meaning both of an individual’s parents must be carriers or affected by the disease to pass the disease on to their offspring. Generally, carriers of the disease have only one functional copy of the SMN1 gene. It is important to note that, in some cases, carriers of the disease have been found to have two copies of the SMN1 gene. These individuals have a duplication of SMN1 on one of their chromosomes and a deletion on the other chromosome. These silent carriers, or (2+0) carriers, have two functional copies of SMN1 but also the potential to give their offspring a chromosome with no SMN1 gene.

With an incidence rate of one in 10,000 live births, approximately 400 new cases of SMA occur every year in the U.S.A. There are multiple subtypes of SMA, and they are defined by the time of disease onset and phenotype of the disease. Details on the different SMA types can be seen in the table below. 

 

SMA type

 

Disease name

Time of onset

Phenotype

SMA Type 0

Not applicable

Birth

Born with a need for mechanical respiratory assistance. Life expectancy is a few weeks.

 

SMAType 1

Werdnig–Hoffmann disease

0–6 months

Disease presents early in life as babies are unable to sit without support. Breathing is also labored and may require mechanical ventilation. Life expectancy varies based on the disease phenotype and can range from a couple years to adolescence, potentially into adulthood.

 

SMA Type 2

Dubowitz syndrome

6-18 months

Affected children are able to sit on their own but are never able to stand or walk. Life expectancy is reduced, but most live into adulthood. 

 

SMA Type 3

Kugelberg–Welander disease

>12 months

Children affected can usually begin to walk without supports but may lose this ability at some stage. Life expectancy is close to normal.

 

SMA Type 4

Not Applicable

Adulthood

Usually sets in after 20 years of age and can affect the extremities of the body, usually requiring use of a wheelchair. Life expectancy is unaffected.

 

 

 

Disease severity can range tremendously as seen in the table above. One of the things that researchers and physicians are looking at from a prognostic value is the number of copies of the highly similar SMN2 gene. The SMN2 gene can make a limited amount of functional SMN protein, but those with only the SMN2 gene cannot produce enough functional SMN protein to fully ward off the disease. The reason the SMN2 gene produces less SMN protein is that the SMN2 gene varies by one nucleotide from the SMN1 gene, which yields a slightly different splicing pattern in the messenger RNA. Instead of all transcripts yielding normal, full-length, and functional SMN protein, SMN2 transcripts usually create degraded SMN proteins with only a small percentage being functional. 

 

  SMN1 vs SMN2 alternate splicing

 

SMN1 SMN2 Splicing Translation

 

Need to determine SMN1 or SMN2 copy numbers as part of your research? Download our Novallele copy number flyer to learn more about our simple and accurate method. 

Stay tuned for the next blog in the series. Next is an update on therapeutic successes and drugs in development for SMA-affected individuals.

 

Products mentioned are for Research Use Only. Not for use in diagnostic procedures.

Nothing herein constitutes medical advice.

Figure used with permission from Butchbach, M. E., & Burghes, A. H. (2004). Perspectives on models of spinal muscular atrophy for drug discovery. Drug Discovery Today: Disease Models1(2), 151-156.

Beginner’s Guide to Mitochondrial DNA Mutations [Free Infographic] | 28 FEB 2018

The mitochondria in our cells are responsible for creating 90% of the energy needed to support our day to day activities. When mutations occur, mitochondria can fail to produce enough energy, which can cause mitochondrial disorders or diseases.

Mitochondrial DNA (mtDNA) is more than just a circle of 37 genes. While there are two copies of nuclear DNA per cell, there are greater than 1000 copies of mtDNA. Mutation rates of mtDNA are 10–17 fold higher compared to those in nuclear DNA, and more than 250 pathogenic mtDNA mutations have been identified.

So how do mitochondrial mutations occur, and what happens when they do? Register to download our free infographic to learn more and explore  detection methods for mutations in the mitochondrial genome.  

 
The GBA Gene — Causing Gaucher Disease and Now Linked to Parkinson's Disease | 27 FEB 2018                  

Gaucher disease, which is caused by mutations in the GBA gene, is more common in the Ashkenazi Jewish population. An interesting link between Gaucher disease and a higher incidence of Parkinson’s disease has been tracked in the past with many hypotheses as to why such a link would exist.

 

One hypothesis is that since mutations in the GBA gene reduce the function of the glucocerebrosidase (GCase) protein, these GBA mutations can lead to a buildup of alpha-synuclein. When alpha-synuclein is present at a high enough concentration, it will clump and turn into Lewy bodies. Lewy bodies are seen in the brain cells of those affected by Parkinson's disease.

Another interesting finding reported by Creese et al. (1) is that GBA mutations can increase the risk of developing more severe disease pathology when it comes to Parkinson’s disease. A meta-analysis in the report uncovered that certain GBA mutations yielded a 2.4-fold increase in dementia, 1.8-fold increase in psychosis, and 2.2-fold increase in depression among patients with Parkinson’s disease. 

Knowing the possible connections of the GBA gene to Parkinson’s disease, pharmaceutical companies have targeted this gene and its effect on the GCase protein. One of these companies is Sanofi with the drug GZ/SAR402671. The drug aims to aid in the breakdown of lipids, including glucosylceramide, which would help prevent buildup of alpha-synuclein, even without a fully-functional GCase protein. 

Want to learn more about disease pathology and the risks associated with common mutations in the APOE and GBA genes? A webinar titled Genetics of Neurodegenerative Disorders - How Mutations Affect Disease Pathology is available on demand. Use the link below to visit the ClinicalOMICS webinar page and register to watch.

 

 

Reference:

1. Creese, B., Bell, E., Johar, I., Francis, P., Ballard, C., & Aarsland, D. (2017). Glucocerebrosidase mutations and neuropsychiatric phenotypes in Parkinson's disease and Lewy body dementias: Review and meta-analyses. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 177(2), 232–241.

Nothing herein constitutes medical advice.

 
5 Tips for Successful CRISPR Screening | 26 FEB 2018

CRISPR technology has revolutionized research, and gene modifications now make it much easier to create cell models. Genotyping using high-resolution melting (HRM) can simplify your workflow by rapidly confirming the creation of single-nucleotide polymorphisms (SNPs) and small indels. Use these five tips to help successfully screen your CRISPR clones:

 

  1. Optimize transfection efficiency so that the maximum number of cells receive Cas9 + gRNA.
    Pro Tip: Use GFP Cas9 to monitor transfection.
  2. Select a fast, easy genotyping technology, such as HRM analysis, suitable or detecting your edit with high specificity.
  3. Confirm successful edits at the first split and dilute.
    Pro Tip: Don’t stop at the first screen if only heterozygous mutants are detected.
  4. Split and dilute wells after subsequent clonal enrichment. Analyze each well using HRM.
  5. Discard all unedited pools.
     

If present, homozygous mutants are detected using HRM. Register to download the full infographic and learn more about screening CRISPR clones.

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