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
• Man-made
– High Molecular
Weight
– Low Molecular
Weight
– Linear
• Nature-made
– High Molecular
Weight
– Low Molecular
Weight
• Man-made
– High Molecular
Weight
– Low Molecular
Weight
– Circular
• Nature-made
– High Molecular
Weight
– Low Molecular
Weight
• RNA
• Nature-made
– High Molecular
Weight
– Low Molecular
Weight
• Man-made
– High Molecular
Weight
– Low Molecular
Weight
Nucleic Acids OMICS
• Genomics
– Various analyses of DNAs
• Qualitative
• Quantitative
• Transcriptomics
– Various analyses of RNAs
• Qualitative
• Quantitative
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. (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
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
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
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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. TemperatureModulated 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
Graphical Scheme
Software
1. HSM (Hitachi) and WAVEMAKER 4.1 (Transgenomic) software
2. Navigator (Transgenomic).
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®
supplied at 1 unit per µl.
System.Discoverase™ dHPLC
DNA Polymerase
is
Equipments
1. Thermocycler (with heated lid).
2. DHPLC WAVE DNA fragment analysis system
(Transgenomic).
3. DNA Sep HT cartridge (cat. no. DNA-99-3710, Transgenomic).
4. Transmit silicone sealing mats (see Note 2) (cat. no. 172024,
Transgenomic).
5. PCR plates (see Note 3) (cat. no. 0030 127307, Eppendorf).
6. Chemical waste container.
Solutions
1. Optimase polymerase (supplied with Mg2+-free buffer and
separate Mg2+ solution)
(cat. no. 703045, Transgenomic).
2. 100 mM dNTP set, PCR grade (cat. no. 10297-117, Invitrogen).
3. Mutation control standards, low-range mutation standard (cat.
no. 560077) and high-range mutation standard (cat. no.
562001, Transgenomic).
4. DNA sizing standard (cat. no. 560078, Transgenomic).
5. Acetonitrile, 99.93+%, HPLC grade (cat. no. 27 071-7, SigmaAldrich).
6. WAVE ion-pairing agent, 2 M triethylammonium acetate
(TEAA) (cat. no. 553303, Transgenomic).
7. Water (e.g., MilliQ water), use 18 M􀀱 resistance or better.
8. WAVE optimized buffers (optional): Buffers A, B, D, and
syringe solution (cat. nos. 553401, 553402, 553404, and 553403,
Transgenomic).
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
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.
Applications
 DHPLC Systems (e.g. WAVE Systems) can be
used for a wide range of applications including:
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Mutation Detection
Sizing
Oligonucleotide Purification and Analysis
Forensics
Microbial Analysis
RNA Isolation and Purification
Single Base Extension
Methylation
With the WAVE System you can:
 Scan fragments for both known and novel mutations/SNPs
without extensive resequencing
 Detect low-abundance mutations in heterogeneous samples
 Enrich for low-abundance alleles
 Fractionate complex mixtures of related fragments based on
differences in sequence content
 Perform targeted mutation/SNP scoring via primer extension
assays
 Analyze end-point products from allele-specific Probe/Primerbased Amplification Technologies
Probe/Primer-based Amplification Technologies
• Primer-based Amplifications:
– Non-Isothermal Amplifications e.g. PCR-based
Methods
– Isothermal Amplifications/Non-PCR-based
Amplifications:
– NASBA
– HDA
– SDA
– , and more than 45 N.A.I.A. Technologies
• Probe-based Amplifications:
– For example: Ligase Chain Reactions
CARTRIDGE dependent Applications e.g. via
WAVE Systems
 DNA-99-3510 DNASep Cartridge 4.6 mm x 50 mm, for variety of DNA
applications including Sizing and Mutation Detection, load capacity of 2µg
 DNA-99-3710 DNASep HT Cartridge 6.5 mm x 37 mm, for variety of DNA
application but especially for Rapid Mutation Detection, load capacity of 4µg
 NUC-99-3550 OligoSep® Cartridge 4.6 mm x 50 mm; for Oligo Purification
work only
 NUC-99-3860 OligoSep Prep HC Cartridge 7.8 mm x 50 mm, for Oligo
Purification, load capacity of 5000µg
 RNA-99-3810 RNASep® Prep Cartridge 7.8 mm x 50 mm, for variety of RNA
applications, load capacity of 50µg total RNA -OR- 1000ng of specific RNA
Chromatograms and pedigree of patients 3 and 16. A. DHPLC analysis in normal
control, patients 3 and 16. Both patients show abnormal DHPLC peak patterns with
two peaks in comparison to normal control. B. Sequencing data in patient 3 and his
family members. C. Sequencing data in patient 16 and his family members.
Example of Oligo Analysis by DHPLC
Thanks for Your Attentions
Reference:
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.