Home graphics: dot Profile graphics: dot Newsletter graphics: dot Distributors graphics: dot Contact graphics: dot Imprint graphics: dot Terms

Newsletter

Direct PCR from FFPE samples, whole blood and barcoding projects

Dear ,

today I would like to send you my best regards from Tokyo; Japan. I'm sitting in front of a big window looking at the skyline from sunny Tokyo (look at the end of this newsletter) and create a newsletter about a new kit developed by Kapa Biosystems for the direct PCR of  tissue samples, FFPE samples and whole blood. We can provide the protocol tds/ , the flier flier/ and 3 different application notes describing the use of this kit for barcoding projects; FFPE samples and processing of whole blood, either EDTA stabilized and/or as dried blood spots appnotes/:

This product is the perfect choice to bundle with the Kapa 2G Robust HS Master Mix and if you ask for our new price list, you will find pricing for the regular kits as well as for combo kits which includes the Extraction kit and the master mix for 100 respectively 500 reactions. A sample kit (KK7150 with both reagents is also available). Furthermore you'll find the pricing for the Library Quantification kits (Next Generation Sequencing) as well as for a new TAQ kit (buffers without MgCl2, but MgCl2 is included in a separate tube). Just send us an email with the keyword pricing mailto:info@nippongenetics.eu and you'll receive the new Excel file promptly.

Rapid and efficient extraction of DNA from a broad range of source material

KAPA Express Extract is a novel thermostable protease and buffer system that allows for the extraction of PCR-ready DNA from various tissue types in as little as 15 minutes. The KAPA Express Extract system has been designed for optimal tissue lysis and DNA preservation. DNA extractions are conveniently performed in a single-tube, without the need for hazardous chemicals and multiple washing steps, thereby greatly reducing the risk of sample loss and contamination. 

Key features of the KAPA Express Extract system include: 

The combination of KAPA Express Extract and KAPA2G Robust HotStart ReadyMix provides the highest performance solution for rapid DNA extraction and consistent downstream amplification. KAPA2G Robust DNA Polymerase is a novel enzyme evolved for increased processivity and exhibits greatly improved tolerance to carryover PCR inhibitors from crude DNA extractions.

Speed: From sample to PCR in less than 15 minutes

 

Versatility: Rapid DNA extraction from a variety of sample types

Traditional methods of DNA extraction are time consuming and laborious or require specialized kits optimized for individual tissue types. KAPA Express Extract kits offer a convenient and efficient alternative for the routine extraction of DNA from a variety of tissue types, including buccal swabs, hair follicles, FFPE tissue, bone marrow, blood, blood spots on denim, and processed foods. KAPA Express Extract kits coupled with KAPA2G Robust HotStart ReadyMix (which contains a novel DNA polymerase tolerant of carryover inhibitors), significantly improves PCR success rates when amplifying from crude extracts.

Figure 2: DNA barcoding is rapidly gaining support as a quick, cost-effective and broadly acceptable tool for species identification. DNA was extracted with KAPA Express Extract from various samples obtained from mammals and fish. From each extract, 2 μl was used directly (without quantification) in a PCR containing KAPA2G Robust HotStart ReadyMix and primers for the ~650 bp cytochrome c oxidase I gene fragment commonly used in species identification (Ivanova et al., 2007). PCR products (10 μl) were analyzed in a 1% agarose gel. Sample origin and type is displayed above the gel.

Reaction products were used directly in standard Sanger sequencing reactions using out-nested M13 primers (2 μl PCR product per 10 μl sequencing reaction). Sequence data was of a high quality and enabled the identification of each species. A section of the sequence trace from Seriola lalandi (Yellowtail amberjack) tissue is presented in the bottom panel.

Fast DNA extraction and increased PCR success rates from FFPE tissue

Figure 3: DNA extracts were prepared from two different FFPE samples using KAPA Express Extract. Sample 1 was archived for >6 months and Sample 2 for >1 year. Each extract was used directly (without quantification) in multiple PCRs containing KAPA2G Robust HotStart ReadyMix and primers for five different fragments (293 bp - 1 kb) of the EGFR gene (corresponding to exons 18 - 21 and 24). Results were compared to those obtained using the same reaction and cycling conditions but using 1 ng purified human genomic DNA as template. With the exception of the 1 kb exon 24 fragment from the older sample, yields and reaction efficiencies were comparable between the FFPE DNA extracts and purified genomic DNA. The PCR products generated from sample 1 were diluted 1:10 and used directly in standard Sanger sequencing reactions. Sequence data (bottom panel, Sample 1 exon 19 fragment) was of a high quality. The mixed sequence starting at the position marked with the arrow confirmed the presence of a 15-nt deletion associated with non-small cell carcinoma diagnosed in the patient from whom Sample 1 was collected.

Routine extraction of DNA from a variety of blood sample types

Figure 4: Extraction and amplification of DNA from different blood sample types for detection of the HLA-B*27 allele. DNA was extracted from 12 human EDTA blood samples with KAPA Express Extract (top panel). 2 μl of each extract was added directly to a 25 μl PCR containing KAPA2G Robust HotStart ReadyMix and two primer sets. The internal control primer set targets a 429 bp fragment of the beta globin gene, whereas the second primer set targets a 141 bp fragment of the HLA-B*27 locus in a sequence-specific manner. Two of the 12 individuals tested positive for the HLA-B*27 allele associated with ankylosing spondylitis. Lanes C- and C+ represent HLA-B*27 negative and positive controls respectively (1 ng purified human genomic DNA as template). DNA was extracted from “Guthrie” cards, FTA cards, or FTA Elute cards (bottom panel) spotted with blood of individuals confirmed to be HLA-B*27 positive (+) or negative (-). DNA extraction and amplification conditions and controls (C- and C+) were the same as for the top panel.

Before I would like to say goodbye, I just want to show you my view over Tokyo. As you might know, I'll stay for two weeks here and I will be back on Monday 08th of February. As you know there is no problem to contact me by email or mobile phone but please keep in mind that we are living in a totally different time zone.Actually we are 8 hours ahead of the middle European time

Thanks for your attention and have a great time

Juergen

No information needed?

Nippon Genetics will focus on the introduction of innovative reagents and instruments for the field of Life Science Research and Diagnostics. If these product are not of interest for you, please send us a blank email  mailto: info@nippongenetics.eu and type unsubscribe in the subject field and we will not send you any further emails.