DECODED
Vol. 2, No. 1
January 2012
The Effect of Genetic
Variation on Phenotype
Genotype to Phenotype
using Next Generation
Sequencing
A Conversation on qPCR
with Jo Vandesompele
Isothermal Assembly for
Easy Gene Construction
Plus regular columns, tips,
and product information
DECODED
January 2012
1
Find Out First
N E W P R O D U C T S & U P D AT E S
More ZEN™/Fluorophore Combos!
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assays can be found on the IDT website, www.idtdna.com,
under the Support menu.
200 pmol Ultramer™ DNA Plate Oligo
IDT proprietary synthesis systems and chemistries allow
high-fidelity synthesis of very long oligonucleotides. These
Ultramer™ oligonucleotides are suitable for demanding applications such as cloning, shRNA, mutagenesis, and gene
construction. The effort and time required for these demanding applications can be greatly reduced by having entire
target fragments synthesized as Ultramers. These oligos have
been available from IDT at 4 and 20 nmole amounts.
Modification Code
(or Name)
2
Features of 200 picomole Ultramer™ DNA Plate Oligos:
•
•
•
Description
/5AmMC12/
5’ amino modifier C12
/5AmMC6/
5’ amino modifier C6
/5Phos/
5’ phosphorylation
/5Biosg/
5’ biotin
/5deoxyI/, /ideoxyI/
5’ or internal deoxyinosine
/5deoxyU/, /ideoxyU/
5’ or internal deoxyruridine
DECODED
For customers who require large numbers of long oligonucleotides synthesized at low scales, IDT now offers 200
picomole Ultramer™ DNA Plate Oligos. This size is ideal for
library production, target capture, and gene construction.
January 2012
•
Available for 45–120 base oligonucleotides
Normalized; dry or resuspended at 2–10 µM in IDTE, pH 7.5.
Sequence fidelity of each Ultramer oligo is established
by electrospray ionization (ESI) mass spectrometry;
traces are accessible in customer accounts on the IDT
website
Supplied in 96- or 384-well plates, enabling automation
and high-throughput techniques
To order 200 picomole Ultramer™ DNA Plate Oligos:
upload or copy and paste your sequences into the plate
configurator on the IDT website at http://www.idtdna.com/
catalog/wellplate/default.aspx. A minimum of 288 oligonucleotides is required per order.
DECODED
Vol. 2, No. 1
January 2012
A Quarterly Journal from Integrated DNA Technologies
Your Research
4
The Effect of Genetic
Variation on Phenotype
8
A Conversation
About qPCR with
Jo Vandesompele
Detecting Mutations Using Targeted Genomic
Enrichment of Multiplex Barcoded Samples
Professor of functional genomics and
applied bioinformatics at Ghent University
Core Concepts
11, 12, 14
Mod Highlight: Photo-Cleavable Spacer
Pipet Tips
18
Steps for a Successful qPCR Experiment
Product Spotlight
20
Up-to-Date Assay Design with
PrimeTime® Pre-designed qPCR Assays
Isothermal Assembly: Quick, Easy
Gene Construction
Methods for Site-Directed Mutagenesis
Competitive Edge
7
Oligonucleotide Quality Requirements
for Mutagenesis Protocols
Find Out First
New Products and Updates
Ask Alex
19
Your Questions For Our Resident Expert
2
Web Tools
Did You Know? 11,16,17,21
Scientific Wisdom
Relocating Your Research to the US
Solutions for Your Research
23
Personalized Group Ordering for Your Lab
DECODED
January 2012
3
Your Research
IDT Products for Innovative Applications
The Effect of Genetic Variation on
Phenotype
Detecting Mutations Using
Targeted Genomic Enrichment
of Multiplex Barcoded Samples
Monitoring Phenotypes Using Next Generation Sequencing
Scientists in Prof Dr Edwin Cuppen’s laboratory (Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and
the University Medical Center Utrecht, both in The Netherlands) use next generation sequencing (NGS) in a systematic
genomics approach to understand the principles by which
genetic variation affects phenotype. Specifically, molecular
phenotypes, like quantitative and qualitative gene expression and epigenetic modifications, are monitored in inbred
animal models for which complete genome sequences are
available. The laboratory also works on human genetics projects driven by the phenotypes presented by patients. Where
a genetic basis for disease is suspected, NGS techniques are
used in an attempt to identify the genetic cause.
Whole Genome Sequencing of Human Samples
Whole genome sequencing can readily be performed on
inbred animals, but it is less straightforward with human
samples for two main reasons:
1) it is relatively expensive as every individual is unique,
and needs to be analyzed independently
2) the majority of information obtained is irrelevant or cannot be interpreted because of our limited knowledge
about genomic elements and the effects on genetic
variation on them.
To overcome some of these hurdles, Prof Dr Cuppen’s group
has spent several years developing techniques for targeted
genomic enrichment (TGE), initially using microarrays and
in-solution methods with DNA or RNA probes. However,
these approaches were not efficient as each assay allowed
enrichment of only a single sample. The group has recently
outlined a more cost-effective method of TGE that enables
multiplexing of samples [1 , 2]. Multiplexing patient samples
speeds up the scientists’ research by enabling examination
of cohorts of patients with the same phenotype, rather than
just single patients.
4
DECODED
January 2012
Targeted Genomic Enrichment Protocol
In their protocol, genomic DNA is isolated from samples
using standard nucleic acid isolation procedures. The DNA is
fragmented by sonication and end-repaired by blunting and
phosphorylation for subsequent ligation to truncated adaptors. For the adaptors, the group uses oligonucleotides manufactured by IDT, which are preannealed before ligating to
the fragmented DNA. The ligated DNA fragments are purified
using AMPure beads (Agencourt). Individual oligonucleotide
barcodes, also manufactured by IDT, are incorporated during
the ligation mediated PCR step to prepare the sequencing
libraries. The barcodes can be introduced by ligation, but the
group chooses to use PCR because it is more flexible and
convenient, and they make use of an obligatory PCR step in
library construction. The barcoded libraries are then pooled
before the researchers perform solution-based enrichment
using Agilent SureSelect technology or microarray-based
capture with custom Agilent SurePrint arrays.
“The quality of IDT oligos is constant, and I do not
know of any situations in which we have experienced problems with specific primers or primer
sets. When experiments fail in our hands we never
doubt the quality of the oligos—we can always be
certain there is another cause. It is typically one of
the other solutions or a problem with the robotics.”—Prof Dr Cuppen, on his laboratory’s experience with IDT oligonucleotides
To prevent hybridization of the long, barcoded 3’ adaptors
to unrelated library molecules, which could reduce enrichment specificity, the scientists use degenerate primers (IDT)
to block barcode and adapter sequences before and during
hybridization-based enrichment. Both methods of enrichment are compatible with Applied Biosystems SOLiD™ 4 NGS
sequencing, which is used in the Cuppen laboratory.
Using Barcode Blockers to Improve Target Read Rate
SOLiD sequencing is performed following clonal amplification of library fragments according to the manufacturer’s
instructions. Using a 10-base barcoding procedure, the
group can perform multiplex amplification of up to 96
independent samples with an average of 59% of reads on
target, which compares favorably with the 60–90% of reads
on target that they routinely obtain for non-multiplexed
enrichment. The use of degenerate barcode-blocking
oligonucleotides that bind to all possible barcode decamers
significantly increases the multiplexed enrichment efficiency,
resulting in an efficient, flexible, and cost-effective means
of targeted genomic enrichment for NGS. This technique is
being applied to the human genetics research performed in
the Cuppen laboratory.
Using Multiplex NGS as a Diagnostics Tool
Prof Dr Cuppen’s human genetics research is focused on the
investigation of genetic variation associated with syndromal
diseases and different types of cancer. Using conventional
sequencing approaches to study syndromal diseases, his
group was limited in the number of disease-related genes
they could investigate, as they could only examine a single
gene per patient at a time. Prioritizing, they would first investigate the most frequently mutated gene. If no mutation
was found, they would move on to the second, and then the
third. This was a lengthy and costly process and, typically, no
more than three genes could be investigated even when
there were other candidate genes. Using the multiplex NGS
protocol they have developed, they are now able to screen
all candidate genes for a given patient simultaneously, in one
assay. Tests can be performed much faster and more comprehensively, resulting in a much shorter wait for patients
eager to get their results.
The group’s goal is to move these multiplex protocols into
routine diagnostic use in order to exploit the immense
Single-plex Enrichment
Multiplex Sequencing
sample 1
sample 2
sample x
enrichment 1
enrichment 1
enrichment 1
benefits gained from NGS. “NGS has revolutionized genomics
research,” says Prof Dr Cuppen, “and it enables faster, cheaper,
and more comprehensive genetic analysis.” However, a higher quality of NGS data is required before NGS can become
a diagnostic tool. Even though good quality data is being
obtained by various laboratories, NGS data quality is generally not as high as that obtained using older, conventional
techniques such as PCR-based dideoxy resequencing, which
is less likely to miscall a base. For this reason, the lab still
performs capillary sequencing as a downstream validation
step to reconfirm findings observed by NGS. Because of the
large amount of sequencing they perform, their validation is
never for just one position, but hundreds that are identified
by NGS. They typically order their IDT oligonucleotides in 96or 384-well plates, which are convenient because these can
be immediately incorporated into the robots that comprise
their automated setups for capillary sequencing, enabling
fast turnaround time from identifying candidate variants to
verifying activating mutations.
Establishing a Personalized Cancer Treatment Center
With other researchers, Prof Dr Cuppen has recently established The Center for Personalized Cancer Treatment (CPCT),
a collaboration of leading scientists from the 3 largest cancer
centers in The Netherlands: UMC Utrecht, the Netherlands
Cancer Institute, and the Erasmus MC/Daniel den Hoed Clinic.
Many cancer drugs that target specific enzymes or pathways
are tested in clinical trial, but most of them won't make it to
Multiplex Enrichment
Multiplex Sequencing
sample 1
sample 2
sample x
add barcode
add barcode
multiplexing
multiplexing
enrichment
sequencing
sequencing
Figure 1. Workflow for Multiplex Barcoding Procedure.
DECODED
January 2012
5
Your Research
IDT Products for Innovative Applications
The Effect of Genetic Variation on
Phenotype
...continued
the market as their effect is only slightly better than existing
drugs. This does, however, not mean that these drugs don't
work. Physicians involved with the center find individual
patients who respond extremely well to drugs that otherwise
do not perform consistently. Thus, some of these drugs can be
effective treatments in specific circumstances.
Researchers at the CPCT believe that cancer drug resistance
can be explained by mutations in the specific signal transduction pathways involved. In a bid to guide treatment
decisions, they have set up a program to sequence up to
2000 candidate genes in biopsies from cancer patients with
metastases in order to add the genetic information of these
metastases to the pathological and radiological analysis of
the patients’ tumors. This research is still in an early experimental phase, and will first be implemented as a way to
stratify patients for specific clinical trials.
According to Prof Dr Cuppen, there are approximately 1000
targeted drugs in early stages of clinical trials, of which only a
few will be brought to market if outdated methods continue
to be used for validation. He hopes the research at the CPCT
will allow them to stratify patient groups to enable selection
of the right patients for treatment with drugs under development. The result will be a wider variety of drugs on the market
and better patient care through increasing the likelihood that
a specific drug will be effective, while avoiding overtreatment
that results from giving patients ineffective therapies. In light
of spiraling healthcare costs, Prof Dr Cuppen states that it is
important to be able to select patients using relatively inexpensive diagnostic tests before administering very expensive
targeted therapies that might be ineffective.
References
1.
Harakalova M, Mokry M, et al. (2011) Multiplexed array-based and insolution genomic enrichment for flexible and cost-effective targeted
next-generation sequencing. Nat Protoc. 6(12):1870-1886.
2.
Nijman IJ, Mokry M, et al. (2010) Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples. Nature Methods
7(11):913–915.
Author: Nicola Brookman-Amissah is a Scientific Writer at IDT.
Prof Dr Edwin Cuppen obtained his PhD at the Radboud University in Nijmegen, The Netherlands. He pursued postdoctoral research at the
Netherlands Cancer Institute in Amsterdam and the Hubrecht Institute in Utrecht, both in the Netherlands. In 2005, Prof Dr Cuppen was
awarded the prestigious European Young Investigator Award for his work on naturally occurring and induced genetic variation in the laboratory rat. He was one of the first researchers to generate gene knock out models in this species. Since 2007 he has been professor of Genome
Biology in the Biology Department of Utrecht University. In 2009 he was appointed professor of Human Genetics and head of the research
section of the Medical Genetics Department of the University Medical Center Utrecht. Pictured here are Prof Dr Edwin Cuppen (back right)
and members of his research group, on retreat in Scotland, UK.
6
DECODED
January 2012
Competitive Edge
T h e I D T A d vantage
Oligonucleotide Quality Requirements for
Mutagenesis Protocols
from four different companies (including IDT). These primer
sets were designed to introduce the following changes:
For mutagenesis applications quality of the oligonucleotide
primers is critical. Impure oligonucleotides can adversely
affect reaction efficiency and can introduce additional,
undesired mutations. IDT monitors every custom synthesis
reaction on every synthesis platform and maintains a basecoupling efficiency that is higher than the industry standard.
IDT has also pioneered the use of high-throughput quality control (QC) methods and is the only oligonucleotide
manufacturer that offers 100% QC and purity guarantees. QC
documents are even made available to customers.
•
Set 1—Single base change C to G (40mers)
•
Set 2—Random 20 bp mutagenesis (60mers)
•
Set 3—Addition of a 20 bp section of the repetitive
element GGT (60mers)
•
Set 4—Deletion of a 20 bp section (60mers)
These oligonucleotides were used in parallel SDM experiments, and resulting clones were screened by IDT scientists.
The data from the cumulative cloning experiments show
that, in every case, using IDT oligonucleotides led to better
mutagenesis results (Table 1).
IDT also evaluates product quality in comparison to competitor products—IDT oligonucleotides consistently rank
as the purest. This exceptional oligonucleotide quality
reduces downstream processing costs, such as assembly
and sequencing, and lowers the overall cost of generating
sequences that carry mutations.
Author: Jaime Sabel is a Scientific Writer at IDT.
In addition to comparing for purity, IDT tests its oligonucleotides against those from competitors in functional studies.
A performance test examined primers used for site-directed
mutagenesis (SDM). Four pairs of SDM primers were ordered
IDT
Competitor I
Competitor O
Competitor G
SDM Set 1
8
7
8
8
SDM Set 2
8
7
8
4
SDM Set 3
8
7
6
2
SDM Set 4
8
7
5
5
% Correct
100%
87.5%
84%
59%
Table 1. Correct Mutants From 8 Colonies Tested for Each Set of Oligos.
Free Mutagenesis Application Guide
The IDT Mutagenesis Application Guide contains an overview of various in vitro mutagenesis approaches, describes the
use of long oligonucleotides called Ultramer™ Oligonucleotides to simplify mutagenesis experiments, gives two protocols for general site-directed mutagenesis, and provides an extensive troubleshooting section. Download in ePub or PDF
format, or request a copy today at www.idtdna.com.
DECODED
January 2012
7
Your Research
IDT Products for Innovative Applications
A Conversation About qPCR with
Jo Vandesompele
thesis research. When he came to me for help
with qPCR data analysis, I explained my data
analysis to him, going through all the formulas
and calculations by hand. He thanked me and
I didn’t see him for a while. When he returned
weeks later, he had programmed all of my
workflows and formulas into Microsoft Excel. I
was really impressed and discussed with him
other things we could do. Six months later, he
came back with what became the first version
of the qbase qPCR data analysis software.
Dr Jo Vandesompele is a professor of functional genomics and applied bioinformatics at Ghent University,
where his lab primarily focuses on cancer genomics.
He is also an internationally recognized expert on
quantitative PCR and co-founder, together with Dr Jan
Hellemans, of the biotechnology company Biogazelle.
Biogazelle offers products and services to assist researchers with all aspects of performing real-time PCR,
from experimental design and data generation through
comprehensive, user-friendly data analysis. We recently
had the opportunity to speak with Dr Vandesompele
about his research, Biogazelle, and the field of qPCR.
How did your experience with qPCR lead to Biogazelle?
I started using qPCR methods in my own research while
working on a rare childhood cancer called neuroblastoma.
I wanted to perform gene expression analysis on some tissue samples, but we only had very small tumor biopsies. A
collaborator suggested that I use quantitative PCR. That was
in 1998 when I still was a graduate student. At that time, I
had not heard of the technology, so I did some research and
quickly realized that it was indeed what we needed since it
is both quantitative and very sensitive because it is PCR. Our
hospital had just purchased one of the first commercially
available real-time PCR instruments, the ABI 7700. I had access to that instrument, and my analysis of those samples
was the start of my experience with qPCR.
Years later, I started working with Biogazelle’s other co-founder, Dr Jan Hellemans, who was employing qPCR for his PhD
8
DECODED
January 2012
Our qbase software was put online and in a
couple of months it had been downloaded
hundreds of times, which led to the suggestion to make qbase into a professional
software package, and shortly after to the
founding of Biogazelle.
What are common problems investigators have when using qPCR?
It really varies a lot with experience and the specific application.
The first problem that many investigators face occurs with
experimental design. Setting up a qPCR experiment is so
simple that it actually becomes dangerous. With the availability of high quality instruments and reagents, it is a very
straight forward process to put samples into a tube, add an
assay and enzyme master mix, and very quickly generate a
lot of data. However, when it is time for data interpretation
the researchers often contact our support and we have to
disappoint them because they didn’t do the right controls to
account for important aspects of the qPCR workflow. There
are probably some who do not think carefully about their
experimental design and proper controls; however, most
people do think about these things, but they just do not
understand all the important issues.
The second challenge, also related to experimental design, is
the selection of suitable reference genes for normalization—
though we addressed this in detail in our seminal paper in
2002 [1]. A common question is whether to use only one
reference gene, or multiple reference genes. Based on our
own data, we recommend the latter. The more basic issue
is how to then use the reference genes to achieve more accurate normalization.
The third common problem is sample quality. Typically, for
gene expression studies using microarrays or RNA sequencing, people do extensive sample quality analysis to avoid
ruining an experiment that can costs thousands of dollars.
However, they often do not perform this analysis for qPCR,
partly because the experiments are relatively cheap, and because qPCR appears very forgiving in terms of sample quality
due to the very small amplicons used for most assays.
some outstanding issues that need to be resolved, with the
major issue being sensitivity and having enough PCR template in these small reaction volumes. However, miniaturization is definitely a trend, and I expect further improvements
in this area.
For example, when studying paraffin-embedded, formalinfixed samples that contain substantially fragmented nucleic
acids, you can relatively easily use qPCR by designing very
small amplicons. The small amplicons make it highly likely
that even degraded samples will have enough of the target
sequence to provide some amplification. And, because
investigators are able to generate product, they believe that
the result is reliable, but this is a major misconception. We
recently published a paper in which we specifically addressed whether RNA quality is an important factor for qPCR
experiments [2]. We methodically determined the degree
of impact that RNA quality has on qPCR experiments and
the ability to accurately analyze the data from those experiments. The results were dramatic and clearly demonstrated
that RNA quality had a direct impact on the variability of
both the selected reference genes, and the measured significance of various biomarkers in more than 600 primary tumor
samples. Unfortunately, many users are still unaware of the
importance of RNA quality.
The other trend I see is digital PCR. Here, reactions contain
approximately one target molecule per reaction. Many reactions must be performed in parallel to accurately determine
what proportion of the total number of reactions contain
template for that target. The individual reactions are not
quantitative, but by doing hundreds or thousands of reactions you can very precisely determine the copy number in
your original sample. So it is extremely powerful, as you can
determine the concentration of your target of interest with
high precision and accuracy.
Much of the training Biogazelle offers through its qPCR
courses is designed specifically to address these important
issues. The courses are very different from those offered by
other companies, which tend to focus on the experimental
procedure itself. We conduct Biogazelle training from two
different perspectives, namely experimental design and
advanced data analysis. The first part of experimental design
involves what is called a “power analysis”, which helps ensure
that researchers have a sufficient number of samples and
controls to enable them to draw statistically significant
results from their analyses. The second part of the training
demonstrates how to set up plates, what controls are needed, and how to use a sample maximization approach versus
an assay maximization approach. We end with biostatistical
interpretation of the results.
What do you see as the future of qPCR methods?
The ongoing trend is towards miniaturization, with increasingly small reactions and qPCR platforms that allow for
several thousand reactions in a single run. All the features
are present to replace microarrays with a technology that is,
in principle, superior with regards to specificity, sensitivity,
turnaround time, accuracy, and dynamic range. There are still
The most important issue with digital PCR is the large
number of reactions you need to perform. In this regard,
platforms that can quantify several thousand reactions at
the same time make digital PCR a very promising technology and it may eventually replace conventional qPCR. Again,
there are some outstanding issues, but for some applications
it definitely has advantages over qPCR today.
What is the significance of the MIQE guidelines?
I am quite happy that there is so much attention given to
the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines [3]. The initiative
to produce the MIQE guidelines was started by Dr Stephen
Bustin, and was the work of an unofficial consortium of qPCR
experts who were all frustrated with the problems with
qPCR methods in published papers. For example, it is very
common that there is not enough information to repeat the
experiment. In addition, investigators do not always address
everything that needs to be accounted for in a proper qPCR
experiment, or you cannot tell if they did because the details
are not sufficiently reported. These issues are critical, as professor Bustin rightly points out, because they actually corrupt
the integrity of the literature with data that is of questionable
quality.
The importance of these concerns in published qPCR studies
compelled us to summarize the most essential criteria that
should be addressed and reported when setting up a qPCR
experiment. The resulting 85 parameter checklist should
help researchers document and perform better qPCR experiments. Investigators can find information on the guidelines
and partners at the MIQE website, www.rdml.org/miqe.php.
DECODED
January 2012
9
Your Research
IDT Products for Innovative Applications
A Conversation About qPCR with
Jo Vandesompele
continued...
We do understand that the guidelines were developed by
academic groups doing research, and that they may not
be appropriate for all fields and applications such as clinical diagnostics, digital PCR, and genotyping. This is why the
consortium needs input from the community, so we can
design extended or modified checklists for specific applications or fields.
How do you think MIQE will affect qPCR reagent suppliers?
MIQE is all about transparency and the ability to replicate
studies. However, there have been extensive discussions in
the consortium about what the minimum requirements for
transparency should be. Based on the original MIQE paper,
one might argue that authors who are using products from
companies that do not provide primer and probe sequences
are not complying with MIQE standards, and the consortium
might want to prevent such companies from selling their
products or promote some vendors over others. In an ideal
world it is probably correct that we should report all of the
relevant experimental information. However, some vendors
have said that they cannot provide all of this information,
and we have to recognize that we live in a commercial environment where we have to find compromise between intellectual property and the ability to replicate an experiment.
This is why we came up with a consensus paper that states
that while it is still recommended to report primer sequences, it is not absolutely required [4]. Instead, the newly modified standard says that providing a context sequence that
can be used to identify the applicable amplicon sequence
+/- 15 bases is sufficient, as long as it allows others to replicate the experiment.
It is interesting to note that many commercial suppliers of
qPCR tools are introducing their products as MIQE compliant.
It demonstrates that these companies see the importance
of what we are trying to accomplish with the guidelines and
want to promote them. With that in mind, it is important to
not give too much weight to suppliers of tools who state
their product is MIQE compliant. It probably means that the
product is a useful tool in the qPCR workflow, but it does not
really add an extensive quality label to that product.
With that in mind, I do appreciate that some qPCR suppliers, like IDT, do provide all the recommended information,
including primer and probe sequences. It is preferable that
companies do that and they should be rewarded somehow
for providing detailed information, at least through appreciation from the scientific community.
10
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January 2012
How has your lab used IDT products and services?
We have been using IDT products since IDT came to Belgium
in 2008. We are great fans of all your primers, and like that IDT
offers quality guarantees, and provides high quality oligos at a
good price. We generally do our own custom designs for our
experiments, and IDT is the preferred supplier for most of our
qPCR primers and synthetic templates. We greatly appreciate
the consistent quality, and speedy delivery, and the fact that
you can accommodate so many scales and delivery options.
We also like having the forward and reverse primers normalized by IDT when we order primers in plates so that we just
need to add water. We can then use our 96-well robotic head
to transfer the assays to experimental plates for qPCR. I think
IDT is the only company that can do this normalization with
consistent quality, at a good price, and with a short turnaround time.
Our lab has had very good experiences working with IDT
and we have essentially converted the whole department to
ordering from IDT.
Do you have any advice for young researchers?
Never give up and always follow your own ideas and dreams.
Scientists should be people with creative minds, always
open to new ideas and adventures. However, many researchers are fairly conservative in their approach to scientific
study. I would encourage young scientists to be as creative
as possible and follow their own gut feeling on how to
design experiments, obviously within the constraints of their
environment and funding possibilities. I think any idea is a
good idea, so follow your own ideas.
References
1.
2.
3.
4.
Vandesompele J, De Preter K, et al. (2002). "Accurate normalization of
real-time quantitative RT-PCR data by geometric averaging of multiple
internal control genes." Genome Biology 3(7): research0034.1–research0034.11.
Vermeulen J, De Preter K, et al. (2011). "Measurable impact of RNA
quality on gene expression results from quantitative PCR." Nucleic
Acids Research 39(9): e63.
Bustin S, Benes V, et al. (2009). "The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments."
Clin Chem 55(4): 611–622.
Bustin SA, Benes V, et al. (2011) Primer Sequence Disclosure: A Clarification of the MIQE Guidelines. Clin Chem 57:919–921.
Author: Hans Packer is a Scientific Writer at IDT.
Core Concepts
S C I E N T I F I C F U N D A M E N TA L S E X P L A I N E D
Modification Highlight: Photo-Cleavable Spacer
Quick Facts:
Availability: DNA or RNA
Scales: 100 nmole to large
scale
Purification: HPLC required
Symbol (for ordering
through IDT): 5’ mod:
/5SpPC/, internal mod:
/iSpPC/
Author: Jeremy Pritchard is a
Technical Support Representative
at IDT.
O
Photo-cleavable (PC) modifications contain a
O
photolabile functional group that is cleavable by
'5
N
H
UV light of specific wavelength (300–350 nm). The
PC spacer is a 10-atom linker arm that can only
NO
be cleaved when exposed to UV light within the
appropriate spectral range. The resulting oligo
O
will have a 5’ phosphate group that is available for
P
O
O
subsequent ligase reactions. PC spacers can be
O
placed between DNA bases or between an oligo
3'
and a terminal modification such as a fluorophore.
IDT also offers 5’ PC Biotin (/5PCBio/), which allows for post-binding release and the design of
controlled capture-and-release assays.
2
-
To order, select PC Spacer or PC Biotin from the 5’ Mods or Internal Mods tab on the oligo
order page. Note that this modification requires HPLC purification.
Disclaimer: PC linkers used in commercial applications require licensing from Ambergen, Inc. (www.ambergen.com).
Did You Know?
Nobel Prize Awards—How
Would You Spend Yours?
PCR is the engine that runs most of molecular biology, and
so its inventor, Kerry Mullis, deserves his Nobel Prize and
the acclaim that comes with it. As you navigate your way
through the countless failures that are inevitable in science,
it may be comforting to remember that even Nobel laureates do not get it right all the time.
SCIENTIFIC WISDOM
Unfortunately the idea was not quite as robust as PCR and
did not work nearly as well. This may be something to consider before you act on your next maverick idea. However, if
you think jewelry containing GeneStones could be successful, the claim to the trademark for the Stargene company
logo has been abandoned, so someone could make use of
the shooting star with the trailing double helix.
One lesson that scientists can learn from Mullis is to stay
out of the fashion industry. In the early 90s he cofounded a
company called Stargene to sell jewelry containing amplified DNA from famous individuals ranging from Mick Jagger
to Abraham Lincoln. If the jewelry was not popular, trading
cards and pens were also considered.
Author: Brendan Owens is Assistant Manager of Technical Support at IDT.
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11
Core Concepts
S C I E N T I F I C F U N D A M E N TA L S E X P L A I N E D
Isothermal Assembly: Quick, Easy Gene Construction
Simplifying Molecular Cloning
Traditional methods of molecular cloning usually involve
multiple enzymatic steps, consisting of separate restriction
digestion, dephosphorylation, and enzymatic ligation reactions. Alternating between the various enzymes and buffer
conditions often requires purification steps, such as gel and
column purification, or ethanol precipitation. These multiple
steps add time and complexity to the protocol, and often result in the loss of DNA. Planning for restriction digest cloning
of multiple fragments can also be time consuming, and designing larger restriction assemblies has additional challenges
as larger constructs will have fewer unique restriction sites.
As with any experimental procedure, it is beneficial to reach
the desired goal in as few steps as possible. This limits the
potential errors due to repeated handling of samples and
reagents, and avoids multiple experimental setups. The isothermal assembly method, recently developed by Gibson, et
al. [1], greatly simplifies the process for molecular cloning of
synthesized DNA molecules. Isothermal assembly also makes
it possible to include larger, more complex assemblies than
traditional cloning methods.
to a typical restriction cloning reaction in which complementary overhangs are no more than 4 bases.
Because the T5 exonuclease processes a random number of
bases before being inactivated there will be single-stranded
gaps between the hybridized 3’ ends and the remaining 5’
ends. The high-fidelity DNA polymerase fills the resulting gaps
and the thermophilic DNA ligase fuses the fragments together.
Design Considerations
A major benefit of the isothermal assembly method is that
it has fewer specific design requirements than other forms
of cloning. For example there is no need to look for endogenous recognition sites for restriction enzymes, or to insert
such sites that could subsequently affect the usefulness of
the final DNA construct. Therefore, the basic principles of
design for isothermal are fairly simple:
•
For the isothermal assembly to work the ends of the
vector and the insert(s) must complement one another.
In general 30 bases of complementarity are optimal at
each end. Ideally the ends should be free from secondary structure.
How Isothermal Assembly Works
•
In a single reaction, isothermal assembly combines several
overlapping DNA fragments to produce a ligated plasmid
ready for transformation. The method relies on use of an enzyme mixture consisting of the mesophilic 5’ T5 exonuclease,
a thermophilic DNA ligase, and a thermophilic proofreading
DNA polymerase. Details of what happens in the reaction are
outlined in Figure 1.
If you are planning to assemble DNA that has been produced from PCR, primers can be designed so that each
primer has 15 bases of overlap into what will be the
adjacent fragment to create the 30 bases of overlap.
•
Since hybridization of long 3’ overhangs is similar to
hybridization of PCR primers, avoid designing 3’ overlaps
in regions with repeated DNA motifs or repeated bases
that can hybridize though improperly aligned.
•
If the final construct is to be linear rather than a fragment subcloned into a plasmid vector, the ends of the
DNA will be retracted at each 5’ end. If desired, these
ends can be filled in a separate reaction using a short
complementary oligonucleotide primer, complementary to the 3’end. This is not necessary for circular constructs, as all gaps are filled and the ends ligated during
the isothermal reaction.
When added to DNA and placed at high temperatures, the
exonuclease digests the double-stranded DNA from its 5’
ends. This reaction is quickly inactivated at 50°C, resulting in
5’ single-stranded ends without completely degrading either
DNA strand. (Because it is a 5’ exonuclease, the T5 activity
does not compete with DNA polymerase and the newly
created overhangs are not immediately filled in when the
exonuclease is inactivated.)
For optimum assembly and ligation, the ends of the DNA
sequences are designed to overlap by at least 30 bases. The
brief 5’ exonuclease activity exposes the overlapping bases,
leaving variable length overhangs at the 3’ ends. These large
overlaps are beneficial for joining fragments as they allow for
highly specific hybridization of DNA fragments—compared
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January 2012
References
1.
Gibson DG, Young L, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5):343–345.
Author: Hans Packer is a Scientific Writer at IDT.
Core Concepts
S C I E N T I F I C F U N D A M E N TA L S E X P L A I N E D
Step 1.
5’
3’
3’
5’
Step 2.
5’
3’
3’
5’
Step 3 and 4.
5’
3’
3’
5’
Step 5.
5’
3’
3’
5’
5’
3’
3’
5’
Figure 1. Isothermal Assembly Method for Double-Stranded DNA. The following events take place in a single 50°C reaction. 1) Individual segments of
dsDNA are designed so that the 3’ strands have complementary overlaps. 2) A mesophilic 5’ exonuclease briefly digests into the 5’ ends of the doublestranded DNA fragments before being inactivated at 50°C. 3) The newly generated complementary 3’ overhangs anneal. 4) A pfu DNA polymerase fills in gaps
completing fragment-containing circular plasmids and leaving free ends retracted on linear fragments. 5) Finally, a thermophilic DNA ligase covalently joins
DNA segments.
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DECODED
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January 2012
13
Core Concepts
S C I E N T I F I C F U N D A M E N TA L S E X P L A I N E D
Methods for Site-Directed Mutagenesis
Site-directed mutagenesis is an in vitro method for creating
a specific mutation in a known sequence, and is typically
performed using PCR-based methods. Primers designed
with mutations can introduce small sequence changes, and
primer extension or inverse PCR can be used to achieve
longer mutant regions. Using these site-directed mutagenesis techniques allows researchers to investigate the
impact of sequence changes or screen a variety of mutants
to determine the optimal sequence for addressing the
question at hand. This article describes simple methods for
site-directed mutagenesis. For a more detailed overview,
see the IDT Mutagenesis Application Guide at
www.idtdna.com.
Traditional PCR
When PCR is used for
site-directed mutagenesis,
the primers are designed
to include the desired
change, which could be
base substitution, addition, or deletion (Figure 1).
During PCR, the mutation
is incorporated into the
amplicon, replacing the
original sequence.
Mutations introduced by
PCR can only be incorporated into regions of
sequence complementary to the primers and
not regions between the
primers [1].
A. Subsitution
B. Deletion
C. Terminal Substitution
Figure 1. Site-Directed Mutagenesis by Traditional PCR. Primers incorporating the desired base changes are used in PCR. As the primers are extended,
the mutation is created in the resulting amplicon.
Inverse PCR enables amplification of a region of
unknown sequence using
primers oriented in the
reverse direction [3]. An
adaptation of this method
can be used to introduce
mutations in previously
cloned sequences. Using
primers incorporating
the desired change, an
entire circular plasmid is
amplified to delete (Figure
3A), change (Figure 3B),
or insert (Figure 3C) the
desired sequence.
References
Site-directed mutagenesis by primer extension involves
incorporating mutagenic primers in independent, nested
PCRs before combining them in the final product [2]. The
reaction requires flanking primers (A and D) complementary
to the ends of the target sequence, and two internal primers
with complementary ends (B and C). These internal primers
contain the desired mutation and will hybridize to the region
to be altered. During the first round of PCR, the AB and CD
fragments are created. These products are mixed for the
second round of PCR using primers A and D. The compleDECODED
To create a deletion, the internal primers, B and C, are positioned at either side of the region to be deleted to prevent it
from being incorporated within fragments AB and CD from
the first round of PCR. The complementary sequences at the
ends of the these fragments, created by primers B and C,
enable hybridization of AB
to CD during the second
round of PCR, and the final
product with the desired
deletion (AD) is created
(Figure 2B).
Inverse PCR
Primer Extension
14
mentary ends of the products hybridize in this second PCR
to create the final product, AD, which contains the mutated
internal sequence (Figure 2A). Longer insertions can be
incorporated by using especially long primers, such as IDT
Ultramer™ oligonucleotides.
January 2012
1.
2.
3.
4.
Zoller MJ (1991) New molecular biology methods for protein engineering. Curr Opin Biotechnol, 2(4): 526–531.
Reikofski J and Tao BY (1992) Polymerase chain reaction (PCR) techniques for site-directed mutagenesis. Biotechnol Adv, 10(4): 535–547.
Ho SN, Hunt HD, Horton RM, et al. (1989) Site-directed mutagenesis by
overlap extension using the polymerase chain reaction. Gene, 77(1):
51–59.
Ochman H, Gerber AS, and Hartl DL (1988) Genetic applications of an
inverse polymerase chain reaction. Genetics, 120(3): 621–623.
Authors: Jaime Sabel and Nicola Brookman-Amissah are Scientific
Writers at IDT.
Core Concepts
S C I E N T I F I C F U N D A M E N TA L S E X P L A I N E D
A. Simple Insertion
B. Deletion
Figure 2. Site-Directed Mutagenesis by Primer Extension. (A) Insertion: Primers B and C contain the complementary sequence that will be
inserted (blue line). Two reactions are performed in the first round of PCR using primer pairs A/B (1) and C/D (2). The resulting amplicons are mixed
with primer pair A/D for the second round of PCR. The complementary ends of the first round amplicons hybridize and the PCR creates the final
product with the desired insertion. (B) Deletion: Primers B and C are located on either side of the sequence to be deleted, and contain sequence
from both sides of the deletion (black or gray additions that match the black or gray original sequence). Two reactions are performed for the first
round of PCR using primer pairs A/B and C/D. The amplicons are mixed with primer pair A/D for the second round of PCR. The overlapping regions
of these amplicons hybridize and the PCR creates the final product with the desired deletion.
A. Deletion
B. Substitution
C. Insertion
Figure 3. Site-Directed Mutagenesis by Inverse PCR. The primers used are 5’-phosphorylated to allow ligation of the amplicon ends after PCR. A
high fidelity DNA polymerase that creates blunt-ended products is used for the PCR to produce a linearized fragment with the desired mutation,
which is then recircularized by intramolecular ligation. (A) Deletion: Primers that hybridize to regions on either side of the area to be deleted are
used. (B) Substitution: One of the primers contains the desired mutation (blue bubble). (C) Insertion: The primers hybridize to regions on either side
of the location of the desired insertion (black, dotted line). One primer contains the additional sequence that will be inserted (blue line).
DECODED
January 2012
15
Core Concepts
S C I E N T I F I C F U N D A M E N TA L S E X P L A I N E D
IDT Product Focus: Reagents for Mutagenesis
Primers
IDT offers custom DNA synthesis on scales from 25 nmole to 10 μmole, and beyond. Every oligonucleotide primer is
deprotected and desalted to remove small molecule impurities. Oligonucleotides are quantified twice by UV spectrophotometry to provide an accurate measure of yield and are quality control checked by mass spectrometry.
Ultramer™ Oligonucleotides
Ultramer Oligonucleotides are 25–200 bases long and are synthesized using IDT proprietary, high-fidelity synthesis systems and chemistries. They are the longest, highest-quality oligonucleotides commercially available and are ideal for demanding applications like cloning, ddRNAi, and gene construction. Researchers can save a great deal of time and trouble
in these applications through direct synthesis of the entire target fragment. Ultramer Oligonucleotides are available on
several scales, and can come with attached modifications such as 5’ phosphate, biotin, and amino modifiers C6 and C12.
Internal degenerate bases, as well as deoxyuracil and deoxyInosine modifications are also available.
Phosphate Modifications
Phosphate modifications may be added to any primer or Ultramer Oligonucleotide. 5’ phosphorylation is necessary if the
product will be used as a substrate for DNA ligase, as when two pieces will be ligated together to create a combined,
longer product. 3’ phosphorylation will inhibit degradation by some 3’-exonucleases and can be used to block extension
by many DNA polymerases.
Genes
IDT provides a confidential custom gene synthesis service. By ordering genes from IDT, researchers not only save money
spent on reagents necessary for construction, cloning, and sequencing, but can also save time by outsourcing the manufacturing of hard-to-clone gene sequences which often results in repeated failures. At IDT, all genes are constructed
using Ultramer Oligonucleotides and the highest fidelity next generation synthesis technology available. Genes arrive in
a plasmid cloning vector and are ready for use in a variety of applications.
For more information and to order these products, please visit IDT’s website at www.idtdna.com.
Did You Know?
ReadyMade Negative
Controls
Introducing foreign RNA via transfection can affect cells in unforeseen ways, such as induction of an immune response in
mammalian cells. Therefore, it is crucial to have an appropri-
16
DECODED
January 2012
S O LU T I O N S F O R Y O U R R E S E A R C H
ate negative control against which one can compare experimental results in gene silencing and functional studies using
DsiRNA duplexes. To this end, IDT has developed a negative
control duplex (DS NC1) that does not target any part of the
human, mouse, or rat transcriptomes. This duplex sequence
serves as an ideal universal negative control for DsiRNA transfections in these organisms. The DS NC1 is available at 1 and 5
nmole scales. To order, go to www.idtdna.com.
Did You Know?
SCIENTIFIC WISDOM
Why are Oligos
Synthesized 3’−5’?
In nature, DNA is formed in the 5’–3’ direction. Early efforts in
DNA synthesis were based on biological synthesis, and thus
the first synthetic oligonucleotides were produced in the
5’–3’ direction. Har Gobind Khorana, a University of Wisconsin biochemist who won the 1968 Nobel Prize in Physiology
or Medicine, led the group that developed the early 5’–3’
synthesis technique using a polystyrene solid support and
three different protecting groups. Though this technique
led to important breakthroughs, it was eventually replaced
in the 1980s by much a more efficient synthesis method
Did You Know?
using phosphoramidite monomers (phosphoramidites are
nucleotides with protection groups which are removed after
synthesis). The growing oligonucleotide is connected to the
solid support, a controlled pore glass bead via the 3’ carbon,
and thus synthesis proceeds in the 3’–5’ direction.
Additional Resources
1.
2.
3.
http://www.idtdna.com/pages/docs/technical-reports/chemicalsynthesis-of-oligonucleotides.pdf
http://www.nobelprize.org/nobel_prizes/medicine/laureates/1968/
khorana-bio.html
Beaucage, S.L.; Caruthers M.H. (1981). "Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide
synthesis". Tetrahedron Letters 22: 1859–1862.
Author: Martin Whitman is a Technical Support Representative at IDT.
SCIENTIFIC WISDOM
The Story of Taq
and Thomas Brock’s
Thermophiles
Convention used to suggest that enzymatic activity would
decrease with an increase in temperature. Imagine Thomas
Brock’s surprise in 1969, when he found bacteria thriving in
the near boiling waters of Yellowstone’s geyser pools. Named
Thermus aquaticus, these bacteria can survive temperatures
between 50°C and 80°C. Various enzymes were isolated
and studied from this organism, but none as significant as
Thermus aquaticus DNA polymerase, better known as Taq
polymerase. The ability of Taq polymerase to survive the high
temperatures required to denature DNA during PCR, meant
that researchers, or rather their students, could perform PCR
without having to add fresh polymerase each cycle. Brock’s
discovery enabled PCR to become an indispensable tool in
genetics and diagnostics, and liberated molecular biology
students everywhere from the burden of babysitting PCR
reactions.
Author: Mehrdad Zarifkar is a Production Scientist at IDT.
Take a class without leaving your computer. View
technical webinars directly from the IDT website.
qPCR Design and Setup
Using SciTools® Web Tools for Oligo Design and Analysis
New topics added regularly. Find them under Support at www.idtdna.com.
DECODED
January 2012
17
Pipet Tips
IDEAS TO STREAMLINE YOUR RESEARCH
Sample Preparation for Successful qPCR
Quantitative PCR (qPCR) enables precise, accurate DNA
quantification. The quality of the starting material is of paramount importance to obtaining reliable, consistent qPCR
data. Here, we provide sample preparation considerations to
help obtain accurate and consistent results when performing 5’ nuclease assays.
•
•
Sample Preparation
The method of isolation depends on the sample type and
experimental conditions. It is important that the sample remains free of nucleases throughout sample preparation and
PCR. Following are recommendations for effective sample
preparation for qPCR.
Avoid Nucleases: RNases and DNases can quickly degrade
samples as well as oligonucleotide primers and probes. They
are ubiquitous and can be difficult to eliminate. Therefore,
precautions must be taken to ensure that samples are protected from these nucleases.
•
•
•
Thoroughly clean sample preparation areas with 10%
bleach solution before you start. Bake glassware and
treat non-RNase-free plastics with a 0.1% solution of
DEPC for 2 hours followed by rinsing with RNase-free
water. Alternatively, IDT provides Nuclease Decontamination Solution, which irreversibly inactivates
nucleases and can be applied to most lab surfaces
(see below, IDT Product Focus: Reagents for Nucleic Acid
Isolation).
Always wash your hands and wear gloves before
handling containers used for RNA sample preparation
and storage.
Use nuclease detection reagents such as RNaseAlert™
and DNaseAlert™ Kits to test your samples and
•
reagents (see below, IDT Product Focus: Reagents for
Nucleic Acid Isolation).
If you find contamination in your samples, replace
all reagents and stock buffers and thoroughly clean
sample preparation areas.
You can add RNase inhibitors to block the action of
some ribonucleases; DNase can be inactivated by
heat treatment.
Regularly decontaminate equipment, such as pipettors and vortex mixers.
Avoid Contamination: Treat your RNA sample with DNase to
prevent amplification of genomic DNA.
RNA Isolation: Be sure to use the isolation method appropriate for your sample type.
•
•
•
•
Use the same isolation and quantification methods for all
samples being assessed in the same experiment.
For maximum recovery, use organic extraction methods such as TRIzol® (Invitrogen) or QIAzol® (Qiagen) for
small RNAs and miRNAs.
For high-throughput processing, use solid phase kits.
Use reagents that are never in contact with potentially contaminating DNA and nucleases by keeping
enzyme mixes, nuclease-free water, primers, probes,
pipettes, tubes, filter tips, and PCR plates away from
areas in which template is present.
RNA Storage: For short-term storage (up to a few weeks),
store RNA resuspended in water or buffer at –20°C. TE buffer
(10 mM Tris, 0.1 mM EDTA) is excellent for this purpose. For
longer term storage, precipitate the RNA (1/10 volume 3
M sodium acetate; 2 volumes 100% ethanol) and store it in
ethanol at –20°C or –80°C. Before use, remember to pellet
and resuspend any RNA that has been stored as a precipitate.
IDT Product Focus: Reagents for Nucleic Acid Isolation
RNase Alert™ and DNase Alert™ Kits—RNase Alert™ and DNase Alert™ Kits enable rapid, sensitive detection of RNases
and DNases. The kits contain fluorescence-quenched probes that emit a fluorescence signal only after nuclease degradation. The signal can be read visually or measured and quantified using fluorometry. Use the assays to test lab reagents,
equipment, and supplies for nuclease contamination, or quantitatively to study enzyme kinetics.
Nuclease Decontamination Solution—Nuclease Decontamination Solution irreversibly inactivates nucleases. It simplifies
sterilizing difficult-to-treat plastic surfaces, and eliminates the need to bake glassware. Just spray, rinse, and let dry.
18
DECODED
January 2012
Ask Alex
YOUR QUESTIONS FOR OUR RESIDENT EXPERT
Why do my OD measurements never match the
values reported by IDT?
What quencher should I use with my qPCR
probes?
OD values are easily affected by a number of small variables.
IDT takes two separate OD measurements to ensure accuracy of the OD calculation. These values are then averaged and
a final OD value is reported. Depending upon the machine
used, the sample prep conditions, and the algorithm applied
to determine the OD values, there will be some variation
from method to method. OD values reported by IDT should
be within 20% of the actual OD value for the product depending upon the preceding variables.
The quencher you select for your qPCR probes will ultimately
depend upon the fluorophore chosen and the real-time
qPCR machine you will be using. Most dark quenchers cover
a relatively large range of wavelengths compared to traditional FRET components. They are typically divided into two
general types: those that quench dyes in the green to yellow
range and those that quench dyes in the orange to red
range. When working with dyes in the green to yellow range,
ZEN™, Iowa Black® FQ, and Black Hole® Quencher-1 are all
appropriate options. When working with dyes in the orange
to red range, Iowa Black® RQ or Black Hole® Quencher-2 are
appropriate. For dyes in the green to yellow range we highly
recommend ZEN™ double-quenched probes as these products contain a second quencher located within the probe sequence itself. This additional quencher ensures that the dye’s
fluorescent signal is quenched to a high degree for greater
signal-to-noise ratios when compared to single-quenched
probes. You can configure your machine for dark quenchers
by selecting black, dark, NFQ, or none as your quencher. This
setting will vary from machine to machine but almost all
qPCR machines are capable of utilizing dark quenchers.
Why do my oligos arrive with different colored
caps?
Cap colors designate products finalized in distinct departments within IDT. Different cap colors are used in the various
purification, specialty production, and chemical synthesis
departments. As such, you will likely receive a variety of
different cap colors as you order different products for your
applications.
What characteristics make up the most efficient
PCR primers?
PCR primer design depends heavily on several factors,
including Tm, length, GC content, secondary structure, and
specificity. A wide range of primer Tms can be used in different PCR applications. While the functional Tm of the primer
sequences will vary, it is important that all primer sequences
within a single reaction have similar Tm values so that they
will anneal properly. Ideally you want the Tm values for your
primers to be +/- 2°C of one another. With regards to length,
primers typically need to be at least 18 bases long in order
to uniquely bind to their target among a heterogeneous
mix of sequences. The length of the primer sequence will
mostly be determined by the desired Tm. GC content is also
important as it will affect Tm and the secondary structures
possible within a given primer sequence. GC percentage
should ideally be between 35− 65%. Strong secondary
structures can prevent a primer from binding to its intended
target. Both hairpins and dimers should be considered when
analyzing secondary structures. Dimer strengths should have
a Delta G value more positive than -9 kcal/mole and software
predicted hairpin Tms should be 5°C below your annealing temperature. Finally, it is recommended to BLAST your
primer sequences to ensure target specificity.
I’ve been using Tsug’s Rule to calculate the Tm of
my oligonucleotide. Is that still the best method
to calculate Tm?
While these rules of thumb offer a general approximation
of Tm, there are better algorithms available that are free and
easy to use, and that will provide an extremely accurate Tm
estimation. We offer one such algorithm for free use in our
SciTools® OligoAnalyzer® application (see www.idtdna.com).
This detailed equation takes into account the effects of sodium and magnesium on Tm calculations. These cations have
a significant impact on the functional Tm of an oligonucleotide in solution due to their charge. For the most accurate
Tm estimation we recommend entering your sequence and
adjusting the ionic concentrations in the OligoAnalyzer tool
so that they match your reaction conditions. You can view
the exact algorithm used for our Tm calculations by clicking
on the Definitions link in the OligoAnalyzer program.
Alex has been busy checking his multiplex qPCR primers to ensure they
do not interact, so Technical Support Specialists Stephanie Youtsey and
Ryan Wilson provided answers. For additional information or answers to
other questions you have, please contact the IDT Technical Support Team.
Contact information can be found on page 22.
DECODED
January 2012
19
Product Spotlight
A P P L I C AT I O N S F O R Y O U R FAV O R I T E P R O D U C T S
qPCR Probes: High Tms Without Sacrificing Quenching
ZEN™ Double-Quenched Probes
For researchers performing qPCR using 5’ nuclease assays,
balancing signal-to-noise and probe specificity can be a challenge. Designing probes in AT-rich regions requires longer
probes to be specific, but choosing probe length can pose
a dilemma. Longer probes provide higher Tms, allowing for
the use of higher melting temperatures, which can prevent
nonspecific probe:target hybridization. However, as probe
length increases, the distance between the fluorophore
reporter and quencher at the probe end increases, resulting
in reduced quenching and thus, poor signal to background
discrimination.
Until recently, Tm enhancers (such as Locked Nucleic Acid
[LNA] bases and Minor Groove Binder [MGB]), or internal
quenchers attached to thymidine bases have been used to
design properly quenched, short probes for qPCR. When
incorporated into the probe, Locked Nucleic Acid (LNA) bases
and Minor Groove Binder (MGB) modifications alter DNA
structure and increase probe stability in duplex formation,
increasing probe melting temperature. Thus, shorter probes
with high melting temperatures and good quenching can
be designed. However, these types of modifications are not
only more costly, but also extremely challenging to design as
melting temperature prediction algorithms for such modifications are not widely known. Also, internal quenchers attached
to thymidine bases require the presence of thymidine residues within the probe sequence.
ZEN Double-Quenched Probes obviate the need for LNAs,
MGB or internal quenchers attached to thymidine bases.
ZEN quencher is an internal quencher that is placed directly
between DNA bases and is used in addition to a 3’ quencher.
It is incorporated at a fixed position, 9 bases from the 5’ end,
which ensures that this quencher is always in close proximity
to the fluorophore. This shortened distance, combined with
the presence of the standard 3’ quencher, allows the design
of longer probes with sufficient melting temperature for
qPCR, without diminishing quencher efficacy. In fact, while
traditional probes do not remain well quenched over 30 bp,
ZEN Double-Quenched Probes maintain a consistently low
background even when longer than 40 bp.
ZEN Double-Quenched Probes can be coupled with several
different fluorescent dyes. See the Find Out First article, More
ZEN™/Fluorophore Combos!, page 2, for details.
MicroRNA Discovery: Solutions for Isolation & Sequencing
miRCat™ Small RNA Cloning Kit
As the regulatory breadth of miRNAs and other small RNAs
becomes more established, many researchers are looking for
tools that will aid in their discovery and isolation. The most
direct approach for identifying these short sequences utilizes
linkers attached to the 5’ and 3’ ends of miRNAs, which allow
for cloning and sequencing. It is widely acknowledged that
cloning efficiency is greatly improved when the linkers are
pre-activated by adenylation [1].
To facilitate the cloning of small RNAs, IDT offers the miRCat™
Small RNA Cloning Kit. The miRCat kit has been specifically
developed to isolate miRNAs in the most efficient manner
possible in order to make the best use of the limited quantities of this precious material.
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January 2012
Further, the miRCat™ cloning kit includes an option to
prepare miRNAs for 454 sequencing and the miRCat-33™
protocol for 5’ ligation–independent small RNA cloning [2],
which is useful for small RNAs without a 5’ phosphate group.
Also available are three ready-made linker oligonucleotides
pre-activated at the 5’ end and blocked at the 3’ end with a
dideoxy-C (ddC), each containing different cloning sites for
increased experimental flexibility.
1. Lau NC, Lim LP, Weinstein EG, and Bartel DP. (2001) An abundant class of
tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science
294:858–862.
2. Pak J and Fire A. (2007) Distinct populations of primary and secondary
effectors during RNAi in C. elegans. Science 315: 241–244
Brendan Owens is Assistant Manager of Technical Support at IDT.
Did You Know?
R E LO C AT I N G Y O U R R E S E A R C H
Paths to Permanent U.S. Residence for Scientists
For many foreign-born scientists, obtaining U.S. permanent
residence is a critical step toward achieving their career objectives. U.S. employers are more willing to hire permanent
residents than nonimmigrant visa holders. Also, many research grant programs are restricted to permanent residents
and U.S. citizens. Scientists seeking permanent residence
typically pursue one of five options:
The EB-1A: The most prestigious path, known as EB-1A, is
for foreign nationals of “extraordinary ability”. It allows selfsponsorship and usually takes approximately six months to
process. The major challenge in pursuing this option is the
high standard for approval. Applicants must prove that they
are within the top 1–2 percent of those in their field. Therefore, EB-1A is typically unsuitable for those in the early stages
of their careers.
The EB-1B: Another relatively rapid path, known as EB-1B,
is open to outstanding researchers and professors. It is
designed primarily for those with tenure-track or permanent
research positions whose prospective employers are willing
to sponsor them. There are some qualifying non-tenure-track
positions at universities and qualifying permanent positions
at for-profit organizations. The major challenge here is the
sponsorship requirement, because many academic institutions are unwilling to sponsor, and industry employers must
meet substantial requirements for research position sponsorship.
National Interest Waiver (NIW): By far the most popular
path for scientists is the NIW program. Since this option falls
within the EB-2 category, where waiting periods for Indian
and mainland Chinese nationals may exceed five years, many
individuals choose to follow the EB-1 route concurrent with
an NIW. The NIW is attractive not only because it permits
self-sponsorship, but also because the standard for approval,
showing “impact” in the field, is not nearly as stringent as the
EB-1A standard. The NIW is suitable for postdoctoral researchers and early-career scientists with a reasonably strong
profile in their field.
Schedule A Group II: A less common path for those with
Bachelor’s or Master’s degrees is referred to as “Schedule A
Group II”. This option does not permit self-sponsorship, and
it requires outstanding, internationally recognized work
within the year prior to filing. In practice, those who meet
the requirements for this category often qualify for an NIW
or an EB-1 visa. However, it may provide an opportunity for a
highly-accomplished individual with a Bachelor’s degree.
PERM Labor Certification: Talented scientists with a more
modest profile in their chosen field may also pursue permanent residence if their employer is willing to file a PERM labor
certification on their behalf. This is accomplished through
a series of steps demonstrating to the U.S. Department of
Labor (DOL) that there are no available and qualified U.S.
workers to fill the position. The major challenges for science
professionals and their employers in this category are the
financial and administrative burdens to the employer and
the length of time required to complete the process.
To learn more about each of these paths to permanent residence, including a detailed analysis of the relevant criteria,
a summary of recent trends, and tips on how to strengthen
your case, see www.immig-chicago.com/Pages/Scientist.
Attorney Elizabeth M. Walder, founder of Immigration Law Associates, P.C., has been
helping foreign-born scientists achieve their immigration objectives for the past
twenty years. Her firm handles the full range of family and employment-based immigration matters, and with science professionals on staff, has developed a unique niche
in the biotech area. Forward your inquiries to [email protected] or visit
www.immig-chicago.com.
DECODED
January 2012
21
Calendar
C ome meet us at t h ese trades h ows
and symposiums
Plant and Animal Genome (PAG)
Jan 14–18, 2012
San Diego, CA
Assembly of Human Genetics
February 2–4, 2012
Marseille, France
Belgian Society of Human Genetics
March 2, 2012
Liege, Belgium
Canadian Society for Biochemistry, Molecular
and Cellular Biology (CSBMCB)
March 15–18, 2012
Whistler, BC, Canada
Association of Biomolecular Research Facilities
(ABRF)
March 17–20, 2012
Orlando, FL
DECODED
A Quarterly Journal from Integrated DNA Technologies
EDITORIAL STAFF
Senior Managing Editors
Nicola Brookman-Amissah,
Hans Packer, Ellen Prediger
Contributors
Erin Bromwell
Ashley Geka
Aurita Menezes
Brendan Owens
Jeremy Pritchard
Martin Whitman
Ryan Wilson
Stephanie Youtsey
Mehrdad Zarifkar
Editorial Review Board
Jess Alexander
Lynette Brown
Stephen Gunstream
Sean McCall
Brendan Owens
Ryan Wilson
Design Director
Tom Malcom
Additional Graphics
Sean Nollen
Subscriptions
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newsletter by registering online at www.idtdna.com/decoded.
Feedback
We hope you find this quarterly newsletter useful and invite you to
provide us with any feedback you have. Send your comments and
suggestions to [email protected].
Customer CARE & Technical Services
American Association of Cancer Research (AACR)
March 31–April 4, 2012
Chicago, IL
Americas
[email protected]
Europe
[email protected]
1-800-328-2661
+32 (0) 16 28 22 60
Other Locations
[email protected]
Analytica
April 17–20, 2012
Munich, Germany
Genomics Research 2012 (Select Biosciences)
April 19–20, 2012
Boston, MA
Experimental Biology
When printed, the DECODED Newsletter is printed
on acid-free, post-consumer recycled paper and
with vegetable-based inks.
www.idtdna.com
©2012 Integrated DNA Technologies, Inc.
April 21–25, 2012
San Diego, CA
Cover: Iced Papyrus. Photo by Tom Malcom.
Knowledge For Growth
PrimeTime® qPCR Assay NOTICE TO PURCHASER: LIMITED LICENSES Use of this product is covered by one or more of the following US patents and corresponding
patent claims outside the US: 5,538,848, 5,723,591, 5,876,930, 6,030,787, 6,258,569, and 5,804,375 (claims 1-12 only). The purchase of this product includes a limited,
non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. Except
under separate license rights available from Applied Biosystems, no right under any other patent claim, or to perform commercial services of any kind, including
without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, or to sublicense, repackage with other products, or
resell in any form, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate
license from Roche. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive,
Foster City, California 94404, USA.
May 24, 2012
Ghent, Belgium
American Society of Microbiology (ASM)
June 16–19, 2012
San Francisco, CA
Click-Chemistry enabled, modified oligos are manufactured and sold under license from Baseclick GmbH, using Baseclick's proprietary Click Chemistry. All such
modified products are sold by IDT for the end-user's internal research purposes only. See www.baseclick.org for further details.
Dicer Substrate. IDT is exclusively licensed under patents owned by the City of Hope and IDT to make and sell DsiRNA Duplex products for use in research and
development. Use of DsiRNA Duplex products or technology in humans or for human or veterinary diagnostic, prophylactic or therapeutic purposes requires a
separate license from City of Hope Medical Center.
TaqMan® is a registered trademark of Roche Molecular Systems that is licensed exclusively to Applied Biosystems Inc. for use in certain non-diagnostics fields.
Black Hole Quencher®, BHQ®, and BHQ-1® are registered trademarks of Biosearch Technologies
The trademarks mentioned herein are the property of Integrated DNA Technologies or their respective owners.
22
DECODED
Volume 2, Number 1
January 2012
Web Tools
Personalized Group Ordering for Your Lab
The LabLinker® web tool is a platform available on the IDT website that lets researchers consolidate orders, customize the lab
ordering process, and share ideas and experimental findings.
The LabLinker web tool allows you to:
•
Consolidate orders to save on shipping charges
•
Access your group's Order History which displays the
sequences previously ordered and offers the option to
reorder sequences
•
Use administrative capability to manage group settings and preferences
•
Invite anyone to join your community: the group is
exclusive to invitees only
•
Set shipping options to provide an automatic, reoccurring order submission schedule, or to approve all
orders prior to submission
•
Use a public discussion board and email notifications
to share information across the group
•
Customize features for….
ʱʱ
Partial or complete shipping
ʱʱ
Addition of end-user initials to sequence
names
ʱʱ
Paper or electronic spec sheets
ʱʱ
Frequency of order submissions
ʱʱ
Photo and information banner on LabLinker
group page
ʱʱ
Countdown time remaining before next
scheduled order placement
To set up a LabLinker account:
1.
Log into your IDT web account, select the
LabLinker® tool from the Order tab at the
top of the screen, and click ‘Create New
Group’.
2.
Fill in the requested information. Include email addresses of lab members who you
would like to join this group. Complete the order submission schedule (next page).
3.
Review the last page which summarizes the settings you selected for your LabLinker group. If the
settings are correct, click ‘Create Group’. Once your
group is created, the members you specified will
receive an invitation email to join the group.
Author: Erin Bromwell is a Customer Support Specialist
at IDT.
DECODED
January 2012
23
1 7 1 0 C O M M E R C I A L PA R K
CORALVILLE, IA 52241
It’s our 25th!
1987 • 25TH ANNIVERSARY • 2012
This year IDT marks our 25th year anniversary of providing high quality scientific products and
services to you, our scientific colleagues. As part of this special occasion, IDT would like to thank
you for your loyalty and support by offering special promotions, events, and educational content
throughout the year. We will be announcing details for these promotions and events in future
issues of our quarterly DECODED newsletter, on our website, and in our regular e-mails. We are
looking forward to an exciting year of celebrating this milestone, and continuing to provide the
quality reagents you have come to trust from IDT.