Pooria Gill PhD of Nanobiotechnology pooriagill@yahoo.com In The Name of Allah DHPLC Principles An Introduction to Denaturing High Performance Liquid Chromatography Pooria Gill PhD of Nanobiotechnology pooriagill@yahoo.com

Nucleic Acids DNA RNA Linear Nature-made Man-made Circular High Molecular Weight RNA Low Molecular Weight Man-made Circular

Nucleic Acids OMICS Genomics Transcriptomics Various analyses of DNAs Qualitative Quantitative Transcriptomics Various analyses of RNAs

Genomics Variations as Mutation/Polymorphism Scanning Procedures for Unknown Mutation Detections: those simple methods which rely on differences in electrophoretic properties being generated between mutant and wild-type nucleic acid by point mutations (these methods cannot, as currently used, detect all mutations, do not localize them within the fragment, and can only be applied to DNA fragments hundreds of bases long). Screening Procedures for Known Mutation Detections: those group which have the potential to detect all mutations. Sequencing is more frequently used to detect unknown mutations than it is for diagnostic purposes.

The Principles for Gene Variation Analysis Bioinformatics Biothermodynamics; Biophysical-Chemistry Spectroscopics Electrophoretics Chromatographics

Scanning Procedures Ribonuclease cleavage (RNAase) Denaturing gradient-gel electrophoresis (DGGE) and related techniques Carbodiimide modification (CDI) Chemical cleavage of mismatch (CCM) Single-strand conformation polymorphism (SSCP) Heteroduplex analysis (HET) Direct sequencing (DS)

Scanning Procedures R.G.H. Cotton. Mutation Research, 285 (1993) 125-144.

Screening Procedures Allele-specific oligonucleotide (ASO) Allele-specific amplification (ASA) Ligation (LIG) Primer extension (PEX) Artificial introduction of restriction sites (AIRS)

Screening Procedures R.G.H. Cotton. Mutation Research, 285 (1993) 125-144.

Heteroduplex analysis (HET) Keen, J. , et al Heteroduplex analysis (HET) Keen, J., et al. (1991) Rapid detection of single base mismatches as heteroduplexes on hydrolink gels, Trends Genet., 7, 5. Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels. Others performed separation of heteroduplexes on normal gels which detected deletions, but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity. Thus far there have been few modifications. The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread, partly because of its later description and partly because of the need for "Hydrolink" gels in the initial description. The main disadvantage is the lack of 100% detection; Like SSCP, the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp).

Technological Improvement of HET to e.g. DHPLC Patent Information#1 Column matrix 1.Bonn, G., Huber, C., Oefner, P. (1994) Verfahren zur Trennung von Nucleinsaeuren. Austrian Patent No. 398 973, Vienna, Austria. 2.Bonn, G., Huber, C., Oefner, P. (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads. U.S. Patent No. 5,585,236. Currently, exclusively licensed to Transgenomic, Inc. Omaha, NE, USA. DHPLC 3.Oefner, P.J., Underhill, P.A. (1998) Detection of Nucleic Acid Heteroduplex Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing. U.S. Patent 5,795,976. [Stanford Reference], [USPTO] 4.Hansen, N.F., Oefner, P.J. (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence. Stanford University Invention Disclosure S97-175. Tangible Research Property in conjunction with U.S. Patent 5,795,976. [Stanford Reference] Currently, licensed to Transgenomic Inc., Omaha, NE, USA; Agilent, Palo Alto, CA, USA; and Varian, Walnut Creek, CA, USA. 5.Oefner, P.J. (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography. U.S. Patent 6,453,244. [Stanford Reference], [USPTO] 6.Huber, C.G., O'Keefe, M., Oberacher, H., Oefner, P.J., Premstaller, A., Xiao, W. Temperature-Modulated Array High-Performance Liquid Chromatography. Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to e.g. DHPLC Patent Information#2 DNA shearing 7.Oefner, P.J., Hunicke-Smith, S. (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides. U.S. Patent 5,846,832. [Stanford Reference], [USPTO] Licensed to Gene Machines, Redwood City, CA, USA DNA markers 8.Oefner, P.J., Underhill, P.A.. Human Y Chromosome Specific Single Nucleotide Polymorphisms. U.S. Patent Pending [Stanford Reference]

Principle of DHPLC Rapid denaturation of DNA by heating, re-annealing by slow cooling. Heteroduplexes form in the presence of two different alleles. DHPLC is a sensitive and specific tool for mutation detection. The first step in DHPLC is PCR amplification of genomic DNA. The amplified DNA fragments are then rapidly denatured by heating, followed by slow cooling to allow re-annealing. Some heteroduplexes will form whenever a mutant allele is present: these contain a region in which the DNA is looped-out around the mismatched region.

DHPLC Graphical Scheme

Nucleic Acid Amplifications e.g. PCR PCR Amplification for Mutation Analysis Using the Transgenomic WAVE® System from Invitrogen Introduction Discoverase™ dHPLC DNA Polymerase is an enzyme mixture composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D polymerase. Pyrococcus species GB-D polymerase possesses a proofreading ability by virtue of its 3’ to 5’ exonuclease activity. Mixture of the proofreading enzyme with Taq DNA polymerase at an optimized ratio increases fidelity approximately eight times over that of Taq DNA polymerase alone and allows amplification of simple and complex DNA templates. The enzyme mixture is provided with an optimized buffer that improves enzyme fidelity. The Discoverase™ dHPLC DNA Polymerase enzyme mixture and buffer formulation have been optimized for use with denaturing high-performance liquid chromatography (dHPLC) systems. They were developed and tested using the Transgenomic WAVE® System.Discoverase™ dHPLC DNA Polymerase is supplied at 1 unit per µl.

Chromatographic Separation Reverse-phase ion-pair system Gradient elution with acetonitrile/water UV detection Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution. Homoduplexes Heteroduplexes Chromatographic separation is achieved using a reverse-phase ion-pair system and a gradient of an organic mobile phase, with UV detection of eluted fragments. The column oven temperature is selected to cause partial denaturation of the marginally less stable heteroduplexes, leading to earlier elution. Thus, up to three additional early-eluting peaks may be observed in the presence of an affected allele. 4 theoretical peaks. TEAA ion-pairing agent. Partially denatured DNA has exposed hydrophobic bases, increasing the partitioning into the organic mobile phase and thereby causing earlier elution.

Results Example of elution profiles for a wild-type and a mutant sample. The mutant sample contains an insertion/deletion mutation in exon 8 of the RET gene. Example of elution profiles obtained for a wild-type and a mutant sample. The mutant sample contains a stop codon mutation in exon 15 of the RET gene.

Thanks for Your Attentions Reference: Methods in Molecular Medicine, Vol. 108: Hypertension: Methods and Protocols, Edited by: J. P. Fennell and A. H. Baker © Humana Press Inc., Totowa, NJ.; Chapter 13.