Navigation Links
Denaturing HPLC (DHPLC)


Most Basic Recommended Cecil Instrument:
Cecil Biocompatible HPLC System 4, plus column oven, plus an optional biocompatible autosampler which will accommodate 96 well plates. A biocompatible installation kit is required. A fraction collector may be added.


Introduction:
DHPLC is an ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) technique.

It identifies mutations and polymorphisms based on detection of heteroduplex formation between mismatched nucleotides in double-stranded PCR amplified DNA. Sequence variation creates a mixed population of heteroduplexes and homoduplexes during reannealing of wild-type and mutant DNA. When this mixed population is analysed by HPLC under partially denaturing temperatures, the heteroduplexes elute from the column earlier than the homoduplexes because of their reduced melting temperature.

Optimisation of DHPLC, requires the generation of a melting profile for each PCR fragment to determine its optimal temperature for analysis.

Usually, the lowest temperature that shows a change in retention time of 1 minute is optimal for identification of sequence variation.


Minimum Analytical Instrument Requirement:

DHPLC requires
• A biocompatible HPLC gradient pumping system with a flow path composed of PEEK tubing fittings, and filters
• An ultraviolet (UV) detector capable of 254 or 260 nm
• A column oven capable of maintaining temperatures of 50°C to 80°C.
• An autosampler configured to handle 2 x 96 0.2-mL microtubes is optimal for automated processing of samples.


An optional fraction collector can be added to the system to automate the isolation and purification of fragments as they elute from the col umn.

There are columns on the market for performing fragment analysis, they include the DNASep Column, the Helix DVB column and the Zorbax Eclipse dsDNA Analysis Column.

The ion-pairing agent used with either column is triethylammonium acetate (TEAA), which mediates binding of DNA to the stationary phase. Acetonitrile is used as an organic agent to achieve subsequent separation of the DNA from the column.


Typical Method:

Using the system described above, the following reagents and column can be used
Standards of ds DNA
• Deonised water
• HPLC grade acetonitrile
• Buffer A, comprising of 100 mM TEAA to pH 7.0, 0.1 mM EDTA,
• Buffer B, comprising of 100 mM TEAA to pH 7.0, 0.1mM EDTA and 25 %v/v acetonitrile
• Helix DVB column, 3mm ID x 50 mm length


Set the detection wavelength to 260 nm.

A typical binary gradient cycle is:
A flow rate of 0.45 ml/minute
Percentage of buffer A, when time is 0.00 minute – 55%
Percentage of buffer A, when time is 0.30 minutes – 50%
Percentage of buffer A, when time is 6.00 minutes – 32%
Percentage of buffer A, when time is 7.00 minutes – 32%
Percentage of buffer A, when time is 7.05 minutes – 55%

Samples and standards may need to be diluted with deionised water.

Run the standard to ensure that the required peaks are well resolved. The column may need to be washed, or a wait may be required for the column to equilibrate to the required temperature.

Between changes in temperature, always ensure that the column has had sufficient time to equilibrate to the required temperature.

Optimisation of the denaturing temperature can be performed by trial or error. Here injection s of the homozygous sample at temperatures from 52 to 67 °C are made. The retention time of the resulting single peak should increase as the temperature is increased. At the denaturing temperature, the retention time will decrease with the increase in temperature.

Optimisation of the DHPLC temperature may also be determined mathematically, using Stanford University’s Melt Calculator http://insertion.stanford.edu/melt.html.

Check for successful PCR amplification by running the homozygous sample under non denaturing conditions , ie less than 50 C and then at the optimal DHPLC temperature. If the PCR conditions have been optimised then a single peak will be seen.

Run the heterozygous sample at the optimum DHPLC temperature. More than one peak should be seen.

If the heterozygous sample does not give a significantly different peak pattern than that seen for the homozygous sample, increase or decrease the column temperature until a different pattern is seen.


Typical Chromatograms:


back to top
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis
2. Detection of K-ras Point Mutations in the Pancreas by Constant Denaturing Gel Electrophoresis Using the DCode System
3. Detection of Mutations in the Fas Antigen of Lymphoma Tumors by RT-PCR and Denaturing Gradient Gel Electrophoresis
4. Detection of Variation in Highly Polymorphic Mhc Genes by Denaturing Gradient Gel Electrophoresis Using the DCode System
5. Detection of p53 Gene in Breast Cancer by Denaturing Gradient Gel Electrophoresis and the DCode System
6. Identification of Disease Causing Mutations in Phenylketonuria by Denaturing Gradient Gel Electrophoresis Using the DCode System
7. Microbial Diversity in Ground and Surface Water Analyzed by Denaturing Gradient Gel Electrophoresis Using the DCode System
8. Separation of HLA-DRB Alleles by Denaturing Gradient Gel Electrophoresis using the DCode System
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:8/17/2017)... CA (PRWEB) , ... August 17, 2017 , ... ... for cancer research and personalized medicine, today announced the launch of a new ... City, Missouri. The study’s goal is to evaluate the potential for early detection ...
(Date:8/16/2017)... , ... August 16, 2017 , ... While art and ... much more closely connected than one might think. A Mesh Is Also a ... at the University City Science Center’s Esther Klein Gallery (EKG) on August 17 and ...
(Date:8/16/2017)... OXFORD, England , Aug. 16, 2017  Kingfisher ... executive search and leadership development, and Virdis Group, global executive ... an exclusive alliance that enables clients to leverage the expertise ... "For our clients here in the Boston ... a diverse population of leadership talent throughout the US, ...
(Date:8/15/2017)... ... August 15, 2017 , ... Pittcon is pleased to once ... scientific instruments. This year’s symposium, organized by the Pittcon 2018 program chair, Annette ... Applications.” This dynamic presentation will discuss novel ionization processes, high throughput IMS-MS technologies, ...
Breaking Biology Technology:
(Date:4/11/2017)... 11, 2017 No two people are ... the New York University Tandon School of Engineering ... found that partial similarities between prints are common ... mobile phones and other electronic devices can be ... vulnerability lies in the fact that fingerprint-based authentication ...
(Date:4/5/2017)... KEY FINDINGS The global market for stem ... 25.76% during the forecast period of 2017-2025. The rise ... growth of the stem cell market. Download ... The global stem cell market is segmented on the ... cell market of the product is segmented into adult ...
(Date:3/30/2017)... March 30, 2017 Trends, opportunities and forecast ... behavioral), by technology (fingerprint, AFIS, iris recognition, facial recognition, ... others), by end use industry (government and law enforcement, ... and banking, and others), and by region ( ... Asia Pacific , and the Rest ...
Breaking Biology News(10 mins):