Navigation Links
Comparison of PCR Kleen Spin Columns to Traditional Methods for Purification of PCR Products Prior to Sequencing, Rev A

Jolanta Kolodziejek, MSc, and Professor Norbert Nowotny, PhD, Clinical Virology Group, Institute of Virology, University of Veterinary Medicine, Vienna , Veterinaerplatz 1, A 1210 Vienna, Austria


Introduction
The purity of the DNA template employed in sequencing PCR (cycle sequencing) is the most critical factor in nucleotide sequencing. The presence of contaminants such as enzymes (nucleases, polymerases), salts, primers, primer- dimers , nucleotides (dNTPs or ddNTPs), ethanol, and others decre ases the quality of the sequencing scans significantly. Primer- or RNA-contaminated samples exhibit a high background and lead to misinterpretation of sequencing data. Contamination with nucleases, salts, nucleotides, and ethanol decreases read length and signal intensity and sometimes results in unre a dable sequence data.

We tested Bio-Rads PCR Kleen spin columns for purification of PCR products prior to sequencing PCR in two different experiments, and compared the resulting nucleotide sequences obtained from these samples with the sequences obtained after purification by traditional methods.


Methods
Experiment 1. Comparison of Effectiveness of Purification Method Prior to Sequencing PCR
A 510 bp RT-PCR product (100 l) was prepared using Borna disease virus as template, and three of four equal-volume aliquots of the PCR product were purified by different methods prior to sequencing. The fourth aliquot was left as an unpurified control.

The first aliquot of the PCR product was purified using PCR Kleen spin columns according to the instructions. The DNA f rom the other two aliquots was recovered from agarose slices using commercial gel extraction kits from two other suppliers (suppliers A and B).

Following purification, PCR aliquots were analyzed on a 1.2% TAE agarose gel and compared with the unpurified PCR p roduct (aliquot 4). The DNA concentration of all three purified PCR products was evaluated spectrophotometrically and compa red with the concentration of the unpurified PCR p roduct. The DNA concentrations were : unpurified product , 0.978 g/l; product purified by PCR Kleen spin column, 0.064 g / l; by supplier A gel extraction kit, 0.016 g/l; by supplier B gel extraction kit, 0.014 g/l.

Following sequencing PCR, the resulting nucleotide sequences were analyzed on an ABI PRISM 310 automated sequencer and compared .

Experiment 2. Sequencing Quality of Longer PCR P roduct After Sequencing PCR and Dye Te rminator Removal
In order to evaluate the effectiveness of PCR Kleen spin columns on the quality of longer nucleotide sequences, thre e aliquots of a 750 bp PCR product derived from Borna disease v i rus were purified using PCR Kleen spin columns. After sequencing PCR, the recovered products were purified for sequencing by three diff e rent methods: We tested one classical purification method (p recipitation in the presence of C2H5O H and 0.5 mM MgCl2) in parallel with two modern commercially available dye removal kits from two other suppliers.


Results
Experiment 1
The best yield of DNA was achieved after purification of the PCR product with the PCR Kleen spin column (see figure).

In contrast, both PCR products obtained by gel extraction, using either the protocol of supplier A or B, were only weakly detected by agarose gel analyses. These results correlated with the amount of DNA in each sample: the PCR product purified with the PCR Kleen column yielded 4 times more of the specific DNA compared to the other samples. Also, the use of PCR Kleen spin columns for purification gave rise to excellent sequencing results (the entire sequence of 510 bp was nicely readable), whereas only 200300 bases were readable in the sequences derived from the other two samples.

Experiment 2
Independently of the purification method used after sequencing PCR (classical or one of the dye removal kits), we achieved the best sequencing results when the PCR products had been purified by PCR Kleen spin columns prior to sequencing PCR: 700 bases were readable, which is the maximum achievable with the ABI PRISM 310 automated sequencer.

Discussion
The results of direct nucleotide sequencing depend on the quality and purity of the PCR products; there fore, the DNA purification step prior to sequencing plays the most importantrole. The purification method used for the sequencing PCR product is of secondary importance .

The use of PCR Kleen spin columns led to excellent sequencing results. Other advantages of this method are:
A simple and rapid procedure (4 min for PCR Kleen column vs. 3 hr for gel purification)

The purified PCR product is immediately available for sequencing PCR

No exposure to ethidium bromide or UV light

It is possible to obtain sequencing results for a PCR product within 1 day

Only one specific PCR product should be present after amplification; in cases where agarose gel electrophoresis exhibits multiple bands, purification methods based on excision of the desired specific band from the gel may be advantageous.

To summarize, we have used PCR Kleen spin columns for purification of many diff e rent PCR products, and have found this method very reliable and superior to other techniques.


The polymerase chain reaction (PCR) process is covered by patents owned by Hoffman-LaRoche. Use of the PCR process requires a license.

ABI PRISM is a trademark of Applied Biosystems.

Info rmation in this tech note was current as of the date of writing (2002) and not necessarily the date this version (rev A, 2003) was published.


back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. PCR Performance Comparisons Between pfuturbo and Taq DNA Polymerases
2. Performance Comparisons of Commercial RT-PCR Systems
3. A Comparison Study of Lipid Tranfection Reagents in A549, NIH 3T3 and COS-7 Cell Lines
4. Comparison of Growth Techniques and Media for the Purpose of Plasmid Isolation from E. coli Using the Eppendorf Perfectprep Plasmid Mini Kit
5. TripleMaster PCR System: Direct Comparison with Competitors
6. Quantitative Measurement of Cell Proliferation Using the BrdU ELISA: A Comparison Between Colorimetric and Chemiluminescent Detection
7. Comparison of different protein quantitation methods
8. Comparison of Different Methods of mRNA Quantitation
9. Bio-Plex Cytokine Immunoassays and ELISA: Comparison of Two Methodologies in Testing Samples From Asthmatic and Healthy Children, Rev A
10. Performance Comparison of the Experion Automated Electrophoresis System and a Competing Automated System for Protein Analysis, Rev A
11. Separation and Comparison of Proteins From Virulent and Nonvirulent Strains of the Fish Pathogen Flavobacterium psychrophilum, Using a 2-D Electrophoretic Approach
Post Your Comments:
(Date:5/1/2015)... Md. and RESEARCH TRIANGLE PARK, N.C. ... UTHR ) announced today that Roger Jeffs , ... provide an overview and update on the company,s business at ... in Boston, Massachusetts . The ... 3:30 PM Eastern Time, and can be accessed via a ...
(Date:4/30/2015)... 2015 /PRNewswire/ - BioAmber Inc. (NYSE: BIOA ), ... it has commenced an underwritten public offering of its ... underwriters a 30-day option to purchase up to an ... offered in the public offering. The offering is subject ... as to whether or when the offering may be ...
(Date:4/30/2015)... SEATTLE , April 30, 2015  The Paul ... Allen Distinguished Investigator (ADI) grants to six groups of ... the most challenging roadblocks in neuroscience: growing mature human ... at a total of $7.5 million over three years.  ... Investigators and their research is especially significant because the ...
(Date:4/30/2015)... SAINT-MALO, FRANCE (PRWEB) April 30, 2015 ... States, and at http://www.lespausa.com , ... a multi-action product combining the benefits of makeup, ... formula. This subtly tinted cream is designed with ... blemishes for an even complexion and a healthy ...
Breaking Biology Technology:BioAmber Inc. Announces Public Offering Of Common Stock 2BioAmber Inc. Announces Public Offering Of Common Stock 3The Paul G. Allen Family Foundation Awards $7.5 Million To Study Brain Cell Growth And Development 2The Paul G. Allen Family Foundation Awards $7.5 Million To Study Brain Cell Growth And Development 3The Paul G. Allen Family Foundation Awards $7.5 Million To Study Brain Cell Growth And Development 4The Paul G. Allen Family Foundation Awards $7.5 Million To Study Brain Cell Growth And Development 5Fleur’s Introduces CC CRÈME Perfect Skin Solution 2Fleur’s Introduces CC CRÈME Perfect Skin Solution 3
... Jolanta Kolodziejek, MSc, and Professor Norbert Nowotny, PhD, Clinical , ... Medicine, , Vienna , Veterinaerplatz 1, A ... Introduction , ... is the most critical factor in nucleotide sequencing. ...
... , , L. A. Knapp, 1, 2 E. Lehmann, 2 ... and D. I. Watkins 2, 3 , ... 2 Wisconsin Regional Primate Research Center, University of Wisconsin, , ... University of , Wisconsin Hospital and Clinics, Madison, Wisconsin, U.S.A. ...
... Menu B. Leddy, Biotechnology Research Department,Orange County ... , Introduction ... of bacterial species from membrane , ... of bacterial cells from membrane biofilms cannot be ...
Cached Biology Technology:Comparison of PCR Kleen Spin Columns to Traditional Methods for Purification of PCR Products Prior to Sequencing, Rev A 2Comparison of PCR Kleen Spin Columns to Traditional Methods for Purification of PCR Products Prior to Sequencing, Rev A 3Comparison of PCR Kleen Spin Columns to Traditional Methods for Purification of PCR Products Prior to Sequencing, Rev A 4Separation of HLA-DRB Alleles by Denaturing Gradient Gel Electrophoresis using the DCode System 2Separation of HLA-DRB Alleles by Denaturing Gradient Gel Electrophoresis using the DCode System 3Separation of HLA-DRB Alleles by Denaturing Gradient Gel Electrophoresis using the DCode System 4Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 2Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 3Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 4Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 5Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 6Separation of 16S rRNA PCR Products From a Model Community Using Temporal Temperature Gradient Gel Electrophoresis, Rev A 7
(Date:4/1/2015)...  NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" or ... growing mobile commerce market, announces the second shipment of ... to early access pre-order customers. Feedback ... outlets including Walmart, Target, AT&T, Dunkin, Donuts, Stop & ... at all outlets and very easy to use. Several ...
(Date:3/31/2015)... -- Elephant Talk Communications Corp. (NYSE MKT: ETAK) ("Elephant Talk" ... Network Architecture (ET Software DNA® 2.0) platforms and cyber ... million for the year ended December 31, 2014 as ... ended December 31, 2013. At the ... multi-year transition away from its legacy landline business to ...
(Date:3/30/2015)... and Markets ( http://www.researchandmarkets.com/research/lc9bnl/global_gesture ) has announced ... in Automotive Sector 2014-2018" report to their offering. ... market in Automotive Sector to grow at a CAGR ... recognition is the ability of a device to identify ... recognition technology can be 2D-based or 3D-based. It uses ...
Breaking Biology News(10 mins):NXT-ID Reports Second Shipment of Wocket Smart Wallets Underway to Early Access Pre-order Customers and Provides User Feedback 2NXT-ID Reports Second Shipment of Wocket Smart Wallets Underway to Early Access Pre-order Customers and Provides User Feedback 3NXT-ID Reports Second Shipment of Wocket Smart Wallets Underway to Early Access Pre-order Customers and Provides User Feedback 4Elephant Talk Reports 2014 Fourth Quarter and Year End Financial Results 2Elephant Talk Reports 2014 Fourth Quarter and Year End Financial Results 3Elephant Talk Reports 2014 Fourth Quarter and Year End Financial Results 4Elephant Talk Reports 2014 Fourth Quarter and Year End Financial Results 5Elephant Talk Reports 2014 Fourth Quarter and Year End Financial Results 6Global Gesture Recognition Market in Automotive Sector Report 2015 - Growing Number of Partnerships 2
... In the last decade, a new strain of MRSA has ... risk of contracting the dangerous bacterial infection. This particular strain ... of genes not shared by any other strains, though it ... to survive and persist in the community. Now, research ...
... The rate of chemical processes in cells is dictated by ... given reaction. Using a versatile method developed at the Institute ... Warsaw, researchers were able to predict for the first time ... . The achievement is important not only for biologists and ...
... the original Otom inhabitants of Xaltocan, the capital of ... long wondered whether they assimilated with the Aztecs or ... research from The University of Texas at Austin, Wichita ... lie in DNA. Following this line of evidence, the ...
Cached Biology News:UNC study may lead to treatments that are effective against all MRSA strains 2Biologistics: How fast do chemical trains move in living cells? 2Biologistics: How fast do chemical trains move in living cells? 3Aztec conquest altered genetics among early Mexico inhabitants, new DNA study shows 2