GENOMICS PRODUCT GUIDE Product Guide.pdf351025 TruePrime® Single Cell Whole Genome Amplification...

GENOMICS PRODUCT GUIDE

Empowering Intelligent Science

Use and Use Restrictions: The Products are sold, and deliverables of any services are provided for the purposes of the buyer’s internal in vitro research, development or educational use only, not for in vivo, or any therapeutic or diagnostic use, nor for resale, or for providing services or any other commercial use of any kind, including without limitation, for any transfer in any form (including as part of a kit) to a third party; for analysis or reverse engineering of the Product or for manufacturing. Products should only be used in accordance with any safety data sheets, guidance or protocols that we issue from time to time and are available for download from our Site. Protective clothing should be used at all times when handling our Products. Safety datasheets relating to all Products are available for download from the Site or upon request. 4basebio grants no other license or rights under any intellectual property in respect of Products or services deliverables and in particular grants no license to use any Product or deliverables for any commercial purposes. Sale of Products or service deliverables by us or our authorised distributors are expressly conditional upon the customer’s agreement with these restrictions, which the customer gives upon placing an order for Products or deliverables. If you wish to use any Product or deliverables for any purpose other than your own internal research as described above, you will require an additional licence from 4basebio. Please contact [email protected].

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IndexTRUEPRIME® DNA AMPLIFICATION

TruePrime® Technology ........................................................................................................................ 4TruePrime® Single Cell Whole Genome Amplification Kit .............................................................. 6TruePrime® Whole Genome Amplification Kit .................................................................................. 8TruePrime® Apoptotic Cell Free DNA Amplification Kit ................................................................... 9TruePrime® Necrotic Cell Free / Exosomal DNA Amplification Kit ............................................. 11TruePrime® Rolling Circle Amplification Kit ..................................................................................... 12

COVCHECKTM GENOME COVERAGE WGA QC KITSCovCheckTM Genome Coverage WGA QC Singleplex Kit ............................................................... 14CovCheckTM Genome Coverage WGA QC Multiplex Kit ................................................................ 15qPCR CovcheckTM Genome Coverage WGA QC Singleplex Kit .................................................. 16

DNA POLYMERASESMagniPhi® Phi29 DNA pol wild type .................................................................................................. 17QualiPhi® Phi29 DNA pol chimera ..................................................................................................... 19

SUNSCRIPT® REVERSE TRANSCRIPTASESunScript® ..............................................................................................................................................22SunScript® Reverse Transcriptase Kit ..............................................................................................24SunScript® One Step RT-PCR Kit .......................................................................................................25SunScript® One Step RT-qPCR Kit .....................................................................................................26

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TruePrime®

Technology

TruePrime® technology is based on the combination of the recently discovered DNA primase TthPrimPol1 and the extremely processive and high fidelity Phi29 DNA polymerase. TthPrimPol is a thermostable member of the recently discovered PrimPol family of enzymes. PrimPols are endowed with primase and polymerase activities. The main novelty of these enzymes is their capacity to synthesize DNA primers. This feature allowed us to develop TruePrime®, a new multiple displacement amplification (MDA) technology based on the combination of TthPrimPol and Phi29 DNA polymerase. As a result, TruePrime® users have a distinct advantage, as TruePrime® does not accept nondenatured DNA strands as templates as readily as random primed MDA reactions. This quality means that any contamination introduced after the denaturation step will not have a strong influence on the composition of the reaction output. We illustrated this unique behaviour through conducting an experiment simulating external DNA contamination by deliberately adding yeast DNA to a denatured human DNA template. The yeast DNA is present in a thousand-fold excess over the target DNA (1pg human genomic DNA).

1 Visit our Nature publication at http://www.nature.com/articles/ncomms13296

1. TthPrimPol binds denatured DNA at different sites

2. TthPrimPol synthesizes short DNA primers

3. Phi29 DNA polymerase displaces TthPrimPol and begins polymerization

4. Phi29 DNA pol performs strand displacement

5. TthPrimPol binds to newly formed DNA and synthesizes new DNA primers

6. Phi29 DNA pol displaces TthPrimPol, binds DNA primers and begins polymerization

TRUEPRIME® IS INSENSITIVE TO EXTERNAL DNA CONTAMINATIONS

TruePrime®Random primers

1 pg of human genomic DNA was denatured and amplified using either TruePrime® (TthPrimPol based MDA) or random primed MDA reactions,. The amplification mix in both cases was purposefully contaminated with 1ng of yeast gDNA (undenatured). Resultant sequencing results showed that TruePrime® preferentially amplified the target human gDNA, as the vast majority of the sequences obtained with TruePrime® were target-derived (human), whereas random primed MDA yielded a majority of contaminant derived sequences (yeast).

The vast majority of sequences obtained using TruePrime® were target derived, whereas random primed MDA yielded predominantly contaminant derived sequences.

TthPrimPol

Phi29 DNA polymerase

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TRUEPRIME®: ABSENCE OF PRIMER ARTIFACTS

TruePrime® Random primers

1pg of human genomic DNA (~1/6 of the content of one human / mammalian cell) was amplified using either TruePrime® (TthPrimPol based MDA) or random primed MDA reactions. Random primed reactions contained 20% ofsequences that cannot be mapped to any organism in sequence databases.

TRUEPRIME® SHOWS AN EXCELLENT GENOME COVERAGE

We have amplified and sequenced the yeast genome using TruePrime® and Illumina technology. Using 2.5 million read pairs we covered 99.4% of the total yeast genome. Shown below is the coverage, illustrating the narrow width of the distribution curve.

Coverage levels within 3 std. dev. from mean

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TruePrime® Single CellWhole Genome Amplification KitWHOLE GENOME AMPLIFICATION

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• Cell lysis buffer included – (If DNA already extracted select TruePrime® WGA)• Primer free method – TthPrimPol synthesizes the primers for Phi29 DNA pol• No primer artefacts – No random extension of primer dimers• Insensitive to external DNA contamination• Works with all types of genomes• Reduced amplification bias in genome coverage – Reliable results• Easy handling in a convenient kit format• Ideally suited for Next Generation Sequencing.

TruePrime® Single Cell Whole Genome Amplification Kit uses a novel and reliable method to achieve accurate genome amplification from single or a few cells. Amplification of a few cells instead of one can improve total coverage breadth if your experimental design allows this.

TruePrime® Single Cell Whole Genome Amplification Kit uses alkaline incubation to allow cell lysis and DNA denaturation of genomic DNA with very low DNA fragmentation. This results in amplified DNA with high integrity and fragment length, ensuring that most of the sequences are uniformly represented.

TRUEPRIME® SHOWS SUPERIOR COVERAGE OF THE GENOME

SAMPLE NONAMPLIFIED DNA TRUEPRIME® (1C) RANDOM PRIMERS COMPETITOR R COMPETITOR G

Mapped reads 99.53% 96.86% 97.42% 99.64% 98.62%

Reads in pane 92.03% 86.77% 59.07% 91.50% 37.94%

Fraction human genome covered 53% 49% 28% 38% 1%

Calculated ideal (Poisson coverage) 59% 57% 44% 59% 31%

Deviation observed from expected 10.2% 13.8% 35.76% 35.4% 96.7%

Single HEK293 cells were amplified with different MDA type single cell amplification methods and coverage compared with the theoretically possible coverage (Poisson) and the coverage in nonamplified DNA from the same HEK293 population. Exactly 12 Mio read pairs were mapped to the human genome.

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CODE DESCRIPTION REACTIONS

351025 TruePrime® Single Cell Whole Genome Amplification Kit (version 2.0) 25 reactions

351100 TruePrime® Single Cell Whole Genome Amplification Kit (version 2.0) 100 reactions

Note: Product Codes 351025 and 351100 have replaced the previous product codes 350025 and 350100 as we are now manufacturing and distributing an improved version of the kit.

Nonamplified DNA

TruePrime®

Competitor R

Competitor G

Random primers

50,000,000 100,000,000 150,000,000

Chromosome 3 coverage(12 Mio read pairs)

Excellent overall mapping data for TruePrime® amplified DNA, closest to nonamplified DNA coverage reached.

Circos plot showing the whole genome coverage for the different methods. Outward to inward: nonamplified DNA, TruePrime®, competitor R, competitor G, generic random primed MDA.

Coverage graph for chromosome three depicting differences in evenness of coverage between the methods. Note the extreme similarity between nonamplified and TruePrime®.

nonamplifiedTruePrime®

Competitor RCompetitor G

Generic RP protocol

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TruePrime® Whole GenomeAmplification Kit

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KEY

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S • Primer free method – TthPrimPol synthesizes the primers for Phi29 DNA polymerase• No primer artefacts – No random extension of primer dimers• Insensitive to external DNA contamination• Works with all types of genomes• Reduced amplification bias in genome coverage – Reliable results• Ideally suited for Next Generation Sequencing.

WHOLE GENOME AMPLIFICATION

The TruePrime® Whole Genome Amplification Kit consists of a set of reagents for multiple displacement amplification (MDA) of genomic DNA from purified material, with a minimal level of bias. The kit uses alkaline incubation to allow DNA denaturation of genomic DNA with very low DNA fragmentation. This results in amplified DNA with high integrity and fragment length, ensuring that most of the sequences are uniformly represented.

Note: Cell lysis buffer not included – Select TruePrime® Single Cell Whole Genome Amplification kit if DNA is not extracted.


370025 TruePrime® Whole Genome Amplification Kit 25 reactions

380100 TruePrime® Whole Genome Amplification Kit 100 reactions

TRUEPRIME® SHOWS EXCELLENT REPRODUCIBILITY

DNA from four single human HEK293 cells was amplified following the TruePrime® protocol for three or six hours, subjected to library prep and sequencing (Illumina HiSeq), and five million read pairs were mapped onto the human genome.

NonamplifiedDNA

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nonamplifiedTruePrime® amplified

single HEK cells

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Left: Shown are comparisons of chromosome 3 and 4 coverage to nonamplified HEK293 cell DNA from the same cells. Note the evenness of coverage, and the high similarity between single cells. Right: Circos plot showing the whole genome coverage of the four replicates (blue=nonamplified; green=amplified).

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TruePrime® ApoptoticCell Free DNA Amplification Kit

FOR THE AMPLIFICATION OF APOPTOTIC CELL FREE DNA DERIVED FROM THE APOPTOSIS CELL DEATH MECHANISM (160–170BP)

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S • Excellent sensitivity – Down to picograms of input material• High amplification yields• Flexibility, up to 150ng or 50µl of cfDNA input• Error free amplification due to the use of high fidelity Phi29 DNA pol• No artefacts derived from random synthetic primers• Streamlined workflow and reduced hands-on time.

One of the main challenges for cell free DNA analysis is the limited amount of template DNA obtained from any bodily fluid, which affects the number and type of tests that can be performed. The TruePrime® Apoptotic Cell Free DNA Amplification Kit addresses this challenge by exponentially amplifying cell free DNA derived from apoptosis cell death mechanism (160–170bp).

The TruePrime® Apoptotic Cell Free DNA Amplification Kit combines the novel TruePrime® DNA amplification technology, with key novel steps of cell free DNA pretreatment, composed of an end repair + dA tailing reaction and ligation of hairpin adaptors, which enables the efficient amplification of apoptotic cell free DNA by TruePrime® following the rolling circle DNA amplification method.

Cell free DNA pretreatment to eliminate protruding ends, restore 5’ phosphates and 3’ hydroxyl groups, and add 3’ dA overhangs. Once the cfDNA is repaired, hairpin adaptors are ligated to both ends of each cfDNA molecule.

DNA sequence and structure of the hairpin adaptor used in the TruePrime® Apoptotic Cell Free DNA Amplification Kit.

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Expected amplification yields

Patients with cancer (N=48) were recruited and 10mls of blood were extracted using Streck Cell Free DNA BCT® tubes. 3mls of plasma was immediately isolated through a double spin centrifugation protocol to avoid genomic DNA contamination from nucleated blood cells. Cell free DNA was purified from 1ml of plasma using Qiagen QIAmp®

Circulating Nucleic Acid Kit. Cell free DNA was quantified using QubitTM. 1ng of each cell free DNA sample was subjected to the TruePrime® apoptotic cell free DNA amplification workflow.

The figure shows the DNA amplification yields obtained in each case. All cell free DNA samples were efficiently amplified, producing enough DNA for any subsequent analysis or technique.

Amplification mechanism of the novel TruePrime® Apoptotic Cell Free DNA Amplification Kit

TthPrimPol generates primers on the hairpin adaptors that are extended by Phi29 DNA pol. The strong strand displacement capacity of Phi29 DNA polymerase allows TthPrimPol to synthesize new primers on the new hairpins generated, resulting in exponential isothermal DNA amplification.


340005 TruePrime® Apoptotic Cell Free DNA Amplification Kit 5 reactions

340020 TruePrime® Apoptotic Cell Free DNA Amplification Kit 20 reactions

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TruePrime® Necrotic Cell Free / Exosomal DNA Amplification KitFOR THE AMPLIFICATION OF GENOMIC DNA STARTING FROM CELL FREE DNA OBTAINED FROM PLASMA, SERUM, URINE OR ANY OTHER BODILY FLUID

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• Excellent sensitivity and reproducibility• Does not generate primer artifacts• Excellent coverage with little bias• Preserves SNVs and SNV frequencies across different sequencing technologies.

The TruePrime® Necrotic Cell Free / Exosomal DNA Amplification Kit uses a novel multiple displacement amplification (MDA) method based on the combination of the recently discovered DNA primase TthPrimPol, and the highly processive and high fidelity Phi29 DNA polymerase, to amplify genomic DNA starting from cell free DNA* obtained from plasma, serum, urine or any other bodily fluid. The strong strand displacement capacity of Phi29 DNA polymerase allows TthPrimPol to generate new primers on the displaced strands that are extended by Phi29 DNA pol, resulting in exponential isothermal DNA amplification.

The TruePrime® mediated amplification will preferentially amplify and therefore select the larger, more cancer specific, DNA fragment size population from the cell free DNA prep. Cell free DNA molecules larger than 1kb can be derived from necrotic cell death or extracellular vesicles like exosomes. This kit provides a method for assessing cancer genome information from all samples, regardless of tumor, with superior sensitivity and specificity. The TruePrime®

Necrotic Cell Free / Exosomal DNA Amplification Kit is reliable at the level of 100pg of cfDNA input, preserving the SNVs and SNVs frequencies across different sequencing technologies and allowing the amplification of cfDNA to levels that permit reliable, high depth sequencing.

TruePrime® Necrotic Cell Free / Exosomal DNA Amplification Kit addresses the issue of liquid biopsy sensitivity and specificity by an adapted amplification of cancer disease specific large fragment cell free DNA.

*Please note this kit cannot be used for the amplification of apoptotic DNA (160–170bp), please see our TruePrime®

Apoptotic Cell Free DNA Amplification Kit.

TruePrime® - amplifiedcell free DNA

Nonamplifiedcell free DNA

Tumor biopsy Tumor biopsy

SNV detection in 50 cancer genes by targeted sequencing. Sigmoid colon undifferentiated adenocarcinoma case (T4a M1a L1 G4).

The SNV correlation is better between TruePrime® amplified cell free DNA and the tumor tissue biopsy than the nonamplified cell free DNA, suggesting that the specific amplification of the large fragment cell free DNA population increases the sensitivity of SNV detection.


330025 TruePrime® Necrotic Cell Free / Exosomal DNA Amplification Kit 25 reactions

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TruePrime® RollingCircle Amplification Kit

The starting material for TruePrime® Rolling Circle Amplification can be:

• Purified DNA: plasmids, cosmids, BACs, M13 clones, etc.• Bacterial cells transformed with plasmids: colonies picked from agar plates, bacterial cultures or glycerol stocks.

SAMPLE AMPLIFICATION

Typical DNA yields from a TruePrime® Rolling Circle Amplification Kit reaction are above 2.5µg per 25µl reaction and three -hour reaction time when starting from 1ng of plasmid DNA (3–10kb). For bigger template molecules (150–200kb) yields are approximately 2µg per 25µl reaction and three-hour reaction time when starting from 10ng. Incubation time can be increased up to six hours if higher amplification yields are required.

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NTC pRSET (3kb)pGEX (5kb) pET28-Liglll (8.4 kb)

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NTC RP11-55A13 (214kb)

RP11-450M16(160kb)

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The TruePrime® Rolling Circle Amplification Kit uses a novel multiple displacement amplification (MDA) method based on the combination of the recently discovered DNA primase TthPrimPol and the highly processive and high fidelity Phi29 DNA polymerase to amplify single or double stranded circular DNA molecules by rolling circle amplification (RCA). The strong strand displacement capacity of Phi29 DNA polymerase allows TthPrimPol to generate new primers on the displaced strands that are extended by Phi29 DNA pol, resulting in exponential isothermal DNA amplification. The TruePrime® Rolling Circle Amplification Kit eliminates the need for overnight cell culture and conventional DNA purification.

AMPLIFICATION FROM BACTERIAL CELLSThe starting material for TruePrime® Rolling Circle Amplification Kit can also be colonies picked from agar plates, bacterial cultures or glycerol stocks:

NTC 2.5μl0

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RCA from glycerol stock 6h 30ºC

RCA from a single colony 6h 30ºC

* Positive control (3kb)

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390100 TruePrime® Rolling Circle Amplification Kit 100 reactions

EXCELLENT SENSITIVITYTruePrime® Rolling Circle Amplification Kit shows an extreme sensitivity down to attograms of input DNA and amplifies a wide range of template sizes:

BENCHMARKINGTruePrime® Rolling Circle Amplification Kit shows the following advantages over current MDA technologies for amplifying circular molecules: Better yields, lower background in the absence of input DNA (NTC) and higher target molecule production (detected by single site digestion).

NTC 100ag 1fg 10fg 100fg 1pg 10pg 100pg 1ng NTC 100ag 1fg 10fg 100fg 1pg 10pg 100pg 1ng

300ng / line. BamHl digestion. Native agarose gel (0.8%) 300ng / line. BamHl digestion. Native agarose gel (0.8%)

pRSET (3kb) pGEX (5kb) pET28-LigIII 8.4kb)

NTC 100ag 1fg 10fg 100fg 1pg 10pg 100pg 1ng NTC 100ag 1fg 10fg 100fg 1pg 10pg 100pg 1ng NTC 1fg 10fg 100fg 1pg 10pg 100pg 1ng 10ng

Plasmid DNA6h 30ºC

M13 ssDNA7.2kb

6h 30ºC

BAC RP11-55A13214kb

6h 30ºC

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CovCheckTM Genome CoverageWGA QC Singleplex Kit

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• Quickly check WGA sample amplification coverage• Only sequence successful amplifications• Ready to use 96 well PCR panel• Avoid long waits and wasted money.

PCR KIT FOR DETERMINING HUMAN SINGLE CELL WHOLE GENOME AMPLIFICATION SUCCESS

4basebio’s CovCheckTM Genome Coverage WGA QC Singleplex Kit provides a ready to use set of endpoint PCR primers in a convenient 96 well plate format, complete with optimized PCR reagents including a premium hot start Taq polymerase. The plate includes four identical sets of 24 different primer pairs that will amplify small portions from each human chromosome, allowing the coverage analysis of four independent single cell whole genome amplifications simultaneously. Easily check results with an agarose gel or any other DNA quantification technique.

Human genome coverage obtained by sequencing versus CovCheckTM Genome Coverage WGA QC Singleplex results. Blue indicates the coverage

obtained by sequencing, marked in red are the chromosomes for which a positive PCR reaction was observed (i.e. an amplicon obtained).

Fraction of genome coverage measured by Illumina sequencing is shown on the y-axis and fraction of positive PCR reactions using CovCheckTM Genome Coverage WGA QC Singleplex Kit is shown on the x-axis. The measures are tightly correlated.

PCR input: 10ng human genomic DNA (male) Promega ref. G147A

PCR input: 10ng of amplified DNA with TruePrime® Single Cell Whole Genome Amplification kit from a single HEK293 cell


CVC0004 CovCheckTM Genome Coverage WGA QC Singleplex Kit 4 reactions

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CovCheckTM Genome CoverageWGA QC Multiplex KitMULTIPLEX PCR KIT FOR DETERMINING HUMAN SINGLE CELL WHOLE GENOME AMPLIFICATION SUCCESS

CovCheckTM Genome Coverage WGA QC Multiplex Kit provides a ready to use set of endpoint PCR primers in a convenient 96 well plate format, complete with optimized PCR reagents including a premium hot start Taq polymerase. The plate includes 16 identical sets of 24 different primer pairs multiplexed in six reations, that will amplify small portions from each human chromosome, allowing the coverage analysis of 16 independent single cell whole genome amplifications simultaneously.

Human genome coverage obtained by sequencing versus CovCheckTM Genome Coverage WGA QC Multiplex results. Blue indicates the coverage

obtained by sequencing. The red sections indicate chromosomes for which a positive PCR reaction was observed (i.e. an amplicon obtained).

96 well plate design containing the six groups of four primer pairs distributed in columns. Each row facilitates the checking of two samples, resulting in 16 samples per plate. Samples are coded with a “C” and a correlative number.

CovCheckTM Genome Coverage WGA QC Multiplex results obtained with human genomic DNA (male and female), and DNA amplified with TruePrime® from single HEK293 cells. Each group (1–6) shows four different amplicons located in four different chromosomes.


CVC000M CovCheckTM Genome Coverage WGA QC Multiplex Kit 16 reactions

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qPCR CovCheckTM GenomeCoverage WGA QC Singleplex Kit

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FOR DETERMINING HUMAN SINGLE CELL WHOLE GENOME AMPLIFICATION SUCCESS

qPCR CovCheckTM Genome Coverage WGA QC Singleplex Kit provides a ready to use set of PCR primers in a convenient 96 well plate format, paired with optimized qPCR reagents. The plate includes four identical sets of 24 different primer pairs that will amplify small portions from each human chromosome, whilst allowing the real time coverage analysis of four independent single cell whole genome amplifications. The optimized qPCR reagents (i.e. qPCR CovCheckTM Genome Coverage WGA QC Singleplex Master Mix: Hot Start Taq DNA polymerase, SYBR Green I, reaction buffer and dNTPs) mean that the qPCR CovCheckTM Genome Coverage WGA QC Singleplex Kit is an easy and convenient approach to getting the best from your single cell experiments, by enabling visualization of specific targets whilst minimizing costs.


CVC1004 qPCR CovCheckTM Genome Coverage WGA QC Singleplex Kit 4 samples

Circos plots showing human genome coverages obtained by sequencing versus CovCheckTM results. Blue indicates the coverage obtained by sequencing. The red sections indicate chromosomes for which a positive PCR reaction was observed (i.e. an amplicon obtained).

Agarose gel electrophoresis of the qPCR CovCheck™ Genome Coverage WGA QC Singleplex amplicons, using single HEK293 cells amplified with TruePrime®.

Chr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

Fraction of genome coverage measured by Illumina sequencing is shown on the y-axis and fraction of positive PCR reactions using CovCheckTM Genome Coverage WGA QC Singleplex Kit is shown on the x-axis. The measures are tightly correlated.

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MagniPhi® Phi29DNA pol wild type

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• Isothermal amplification

• High precision amplification – Up to 100 times less error prone than Taq DNA polymerase

• Excellent reproducibility

• Minimal required template – Perfect for rare / hard to come by samples

• High yielding enzyme – Higher amplification product yield in Whole Genome Amplification and Rolling Circle Amplification reactions compared to PCR based technologies.

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DNA AMPLIFICATION

MagniPhi® Phi29 DNA pol wild type is the wild type Phi29 DNA polymerase currently in use in the TruePrime® DNA amplification kits as supplied by 4basebio. The highly processive capability of the enzyme (up to more than 70kb per binding event) paired with a strong strand displacement capacity makes MagniPhi® Phi29 DNA pol wild type the perfect choice for isothermal DNA amplification. The 3’ --> 5’ proofreading exonuclease activity of the MagniPhi® Phi29 DNA pol wild type means that MagniPhi® Phi29 DNA pol wild type has a very high fidelity of synthesis, resulting in up to 100 times less errors than using Taq polymerase, while the amplification reaction yields more Whole Genome Amplification (WGA) product compared to PCR based techniques. These distinctive features make this enzyme the perfect choice for isothermal DNA amplification or any application requiring a high fidelity DNA polymerase with strong strand displacement capacity.

The graph above (Figure 1.) shows the high sensitivity and amplification efficiency of MagniPhi® Phi29 DNA pol wild type at varying template DNA concentrations. As illustrated, MagniPhi® Phi29 DNA pol wild type shows high sensitivity and yield, even at very low template DNA concentrations.

MAGNIPHI® PHI29 DNA POL WILD TYPE SHOWS HIGH SENSITIVITY AND AMPLIFICATION YIELD IN WHOLE GENOME AMPLIFICATION (WGA) REACTIONS

Figure 1: MagniPhi® Phi29 DNA pol wild type amplification yield using random synthetic primers in WGA reactions

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500025 MagniPhi® Phi29 DNA pol wild type 25 reactions

500100 MagniPhi® Phi29 DNA pol wild type 100 reactions

MAGNIPHI® PHI29 DNA POL WILD TYPE SHOWS HIGH SENSITIVITY AND AMPLIFICATION YIELD WHEN USED IN ROLLING CIRCLE AMPLIFICATION (RCA) REACTIONS

As shown in the graph above (Figure 2.), the high fidelity and strong strand displacement efficiency of MagniPhi® Phi29 DNA pol wild type is well suited in the use of RCA reactions, with high yields produced from very limited concentrations of template DNA.

Figure 2: MagniPhi® Phi29 DNA pol wild type reaction using random synthetic primers when used in RCA reactions

As can be seen in Figure 3 above, MagniPhi® Phi29 DNA pol wild type was benchmarked against two of the leading market Phi29 DNA polymerase providers, comparing the resulting DNA yield between the three products, with varying template amounts in WGA reactions.

Figure 3: MagniPhi® Phi29 DNA pol wild type benchmarking using random synthetic primers against leading market competitors when used in WGA reactions

M13 ssDNA input

MagniPhi® Phi29 DNA pol wild type

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QualiPhi® Phi29DNA pol chimera

• Isothermal amplification

• High precision amplification – Up to 100 times less error prone than Taq DNA polymerase

• Excellent reproducibility

• Minimal required template – Perfect for rare / hard to come by samples

• High yielding enzyme – Higher amplification product yield compared to conventional MDA technology in Whole Genome and Rolling Circle Amplification reactions.

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QualiPhi® Phi29 DNA pol chimera is a novel, highly processive chimeric form of Phi29 DNA polymerase, expertly engineered for enhanced sensitivity and efficiency (de Vega et al., 2010).

As the chimeric version of the wild type Phi29 DNA polymerase, QualiPhi® Phi29 DNA pol chimera has preserved the high processivity – strong strand displacement capacity and the excellent 3’ --> 5’ proofreading exonuclease activity resulting in very high synthesis fidelity – of the wild type Phi29 DNA polymerase. QualiPhi® Phi29 DNA pol chimera has been engineered to dramatically increase its sensitivity, thus enabling it to more efficiently amplify DNA from low input concentrations. With enhancements furthermore resulting in yields far exceeding wild type Phi29 DNA polymerases, QualiPhi® Phi29 DNA pol chimera is a premium polymerase giving researchers the opportunity to combine primers with a specialist polymerase enhanced for work with low concentration template DNA.

The graph above (Figure 1.), shows the high sensitivity and amplification efficiency of QualiPhi® Phi29 DNA pol chimera at varying template DNA concentrations. As depicted, QualiPhi® Phi29 DNA pol chimera shows exceptionally high sensitivity and yield, even at very low template DNA concentrations.

QUALIPHI® PHI29 DNA POL CHIMERA SHOWS HIGH SENSITIVITY AND AMPLIFICATION YIELD IN WHOLE GENOME AMPLIFICATION (WGA) REACTIONS

Figure 1: QualiPhi® Phi29 DNA pol chimera amplification yields using random synthetic primers in WGA reactions

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QualiPhi® Phi29 DNA pol chimera was benchmarked against two of the leading market Phi29 DNA polymerase providers, comparing the resulting DNA yield between the three products, with varying template amounts in WGA reactions. As can be seen in the resulting graph (Figure 3.), neither of the leading market competitors are able to compare to the efficiency and yield of the QuaiPhi® Phi29 DNA pol chimera.

Figure 3: QualiPhi® benchmarking using random synthetic primers against leading market competitors in WGA

QUALIPHI® PHI29 DNA POL CHIMERA SHOWS SUPERIOR DNA YIELD IN WHOLE GENOME AMPLIFICATION (WGA) REACTIONS COMPARED TO LEADING MARKET COMPETITORS

As shown in the graph above (Figure 2.), the high fidelity and strong strand displacement efficiency of QualiPhi® Phi29 DNA pol chimera is ideally suited for use in RCA reactions, with very high yields produced from very limited concentrations of template DNA.

QUALIPHI® PHI29 DNA POL CHIMERA SHOWS HIGH SENSITIVITY AND AMPLIFICATION YIELD WHEN USED IN ROLLING CIRCLE AMPLIFICATION (RCA) REACTIONS

Figure 2: QualiPhi® Phi29 DNA pol chimera reaction yield using using random synthetic primers when used in RCA reactions

M13 ssDNA input

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510025 QualiPhi® Phi29 DNA pol chimera 25 reactions

510100 QualiPhi® Phi29 DNA pol chimera 100 reactionsDN

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QUALIPHI® SHOWS SUPERIOR DNA YIELD IN RCA REACTIONS COMPARED TO LEADING MARKET COMPETITORS

de Vega, M., Lazaro, J.M., Mencia, M., Blanco, L., Salas, M., 2010. Improvement of Φ29 DNA polymerase amplification performance by fusion of DNA binding motifs. PNAS, 107(38), 16506-16511.

References

QualiPhi® Phi29 DNA pol chimera was benchmarked against two of the leading market competitors’ products, investigating the resulting DNA yield between the three products with varying template volumes in RCA reactions. As is clearly shown (see Figure 4.), QualiPhi® Phi29 DNA pol chimera shows marked superiority in yield, even at femtograms (10-15 g) of input template DNA.

Figure 4: QualiPhi® Phi29 DNA pol chimera benchmarking using random synthetic primers against leading market competitors in RCA

Figure 5: QualiPhi® Phi29 DNA pol chimera Kit

M13 ssDNA input

QualiPhi® Phi29 DNA pol chimera

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BASIS OF THE REVERSE TRANSCRIPTION REACTION

The oligos are hybridized to the RNA strand

The reverse transcriptase binds and synthesizes the complementary cDNA strand

The reverse transcriptase is inactivated

SUNSCRIPT® REVERSE TRANSCRIPTASE ALLOWS REACTION TEMPERATURES UP TO 85ºC

100ng of total RNA from HeLa cells were reverse transcribed at the indicated temperatures for 60 minutes with SunScript® Reverse Transcriptase and Oligo(dT)18 primer, followed by inactivation at 95°C for ten minutes. Presence of GAPDH cDNA was detected by PCR (product length 305bp).

High reaction temperatures are advantageous in facilitating the transcription of RNA with complicated secondary structures.

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SUNSCRIPT® REVERSE TRANSCRIPTASE: HIGH SENSITIVITY OVER A WIDE RANGE OF RNA CONCENTRATIONS

Sensitivity test: RT reactions were performed at 70°C for 60 minutes. Inactivation at 95°C for ten minutes

Primer set: Human GAPDH305bp set

1- 1µg Human total RNA from HeLa cells

2- 100ng Human total RNA from HeLa cells



5- 100pg Human total RNA from HeLa cells

SUNSCRIPT® REVERSE TRANSCRIPTASE: TRANSCRIPTION OF LONG mRNAs

Assay for long mRNA reverse transcription: Three independent RT reactions were performed at 65°C for 60 minutes using Oligo(dT)20 primers. Inactivation at 95°C for ten minutes. PCR Primer sets: mmNEB (>16kb from 3’end of mRNA; mRNA total length 22.5kb); mmDnah8 (>8.5kb from 3’end of mRNA; mRNA total length 14.5kb).

PCR product

AAAAAAT T T T T T

mmNebulin mRNA

16.7kb22.5kbPCR product

AAAAAAT T T T T T

mmDnah8 mRNA8.5kb14.5kb

1- SunScript® RT RNaseH- replicate 1



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SUNSCRIPT® REVERSE TRANSCRIPTASE: HIGH cDNA SYNTHESIS RATE

PCR Primer set: hsHERC4033 (>11kb from 3’end of mRNA; mRNA total length 15.3kb)

1µg of total RNA from HeLa cells was reverse transcribed at 65°C for the indicated times with SunScript® Reverse Transcriptase and Oligo(dT)18 primer, followed by inactivation at 95°C ten minutes.

SunScript® can completely synthesize cDNA of up to 11kb in length in only five minutes.

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SunScript® ReverseTranscriptase Kit

SunScript® Reverse Transcriptase Kit is a novel, highly thermostable reverse transcriptase engineered from the well characterized HIV-1 RT. It allows reaction temperatures up to 85°C, has RNA and DNA dependent polymerase activity and offers improved efficiency of cDNA synthesis. The elimination of RNaseH activity provides this enzyme with the best performance to allow high yields of full length cDNA synthesis on long RNA molecules with complex secondary structures.

Through lyophilization, the SunScript® Reverse Transcriptase is preserved in such a manner as to maintain full integrity while making the enzyme more robust, allowing ambient temperature shipping. This greatly reduces cost and ease of transport.

These kits include 5X Reaction Buffer and 0.1M DTT. Contents are provided in clear, screw cap tubes with color coded lids for quick reference during experiments. SunScript® Reverse Transcriptase Kit has been tested to be free of endonucleases, exonucleases and ribonucleases, with the purity of the enzyme guaranteed to be above 95%.

• Highly thermostable: active up to 85ºC to resolve even the most complex RNA structures

• High reaction speed of more than 30 bases per second

• High yields of first strand cDNA

• Complete reverse transcription of very long RNA molecules

• High sensitivity (down to picograms of starting total RNA)

• Yields highly consistent and reproducible results.KEY

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421010 SunScript® Reverse Transcriptase Kit 10 reactions



424008 Lyophilized SunScript® Reverse Transcriptase Kit 8 reactions



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SunScript® One Step RT-PCR Kit

OVERVIEW OVER THE ONE-STEP RT-PCR REACTION

100 ng of total RNA from HeLa cells were reverse transcribed and amplified with SunScript® One Step RT-PCR Kit and specific primers for the indicated genes (four replicates are performed per gene). The four independent replicates per gene show similar sensitivity and yield, making this kit the best choice for processing multiple samples with minimum deviations.


430025 SunScript® One Step RT-PCR Kit 25 reactions

430100 SunScript® One Step RT-PCR Kit 100 reactions

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• Fast, specific and sensitive system for endpoint RT-PCR reactions

• Performs both reverse transcription and PCR in the same single tube

• Allows “one step reaction” to minimize contamination

• Convenient system to process multiple samples

• Based on SunScript®, it is an excellent choice to amplify complex RNAs

• High reproducibility of reactions

• Robust and reliable.

SunScript® One Step RT-PCR Kit is an easy and reliable system designed for fast, specific and sensitive endpoint RT-PCR reactions. The kit contains all the components needed to perform both reverse transcription and PCR amplification in the same single tube by using gene specific primers, in a “one step reaction”. This minimizes contamination and constitutes a convenient system to process multiple samples.

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SunScript® One Step RT-qPCR KitSunScript® One Step RT-qPCR Kit is an easy and reliable system designed for fast RT-qPCR (real time) reactions based on detection of double stranded DNA binding dyes. One key advantage of the kit is the elevated temperature for performing the RT step thus allowing higher specificity of the reaction, and a higher likelihood in obtaining reaction products for difficult targets. Moreover, the excellent kinetic performance of the SunScript® Reverse Transcriptase allows for speed optimization of the protocol.

The kit contains all the components (including SYBR Green I, a fluorescent DNA binding dye) needed to perform both reverse transcription and qPCR reaction in the same reaction by using gene specific primers in a one step reaction.

Comparison of amplification of the low abundance transcripts BAX and HERC2 at 50ºC and 60ºC reverse trancription reaction temperature. RNA source: HEK293. Total RNA: 100ng. Light blue: SYGNIS SunScript® One Step RT-qPCR Kit, dark blue, red, orange: Competitor kits.


450100 SunScript® One Step RT-qPCR Kit 100 reactions

• Superior performance in comparison to all tested competing high temperature RT-qPCR kits

• One Step RT-qPCR Kit with the highest temperature RT step on the market (up to 75ºC)

• Fast RT-qPCR protocol possible due to high synthesis rate of SunScript® Reverse Transcriptase and our high-quality Taq Polymerase

• Broad dynamic range with high sensitivity for low abundance transcripts

• High reproducibility of reactions

• Easy handling: Kit contains all reagents in an easy-to-use format

• Kit is compatible with all qPCR machines allowing quantification by dye intercalation (e.g. Bio-Rad CFX series, Roche LightCycler 480 series). Kit is optimized for plate based machines.

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GENOMICS PRODUCT GUIDE Product Guide.pdf351025 TruePrime® Single Cell Whole Genome Amplification...

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