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

2. Nucleic Acids DNA –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 Man-made –High Molecular Weight –Low Molecular Weight RNA Nature-made –High Molecular Weight –Low Molecular Weight Man-made –High Molecular Weight –Low Molecular Weight

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

4. Genomics Variations as Mutation/Polymorphism Scanning Procedures for Unknown Mutation Detections: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: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.

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

6. 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)

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

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

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

10. Heteroduplex analysis (HET) Keen, J., et al. (1991) Rapid detection of single base mismatches as heteroduplexes on hydrolink gels, Trends Genet., 7, 5. containing single base-pair mismatches heteroduplexes on nondenaturing gelsHeteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels. on normal gels deletionsnot detect point mutationslesser sensitivityOthers 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. main advantage simplicity HydrolinkThus 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. disadvantagelack of 100% detection fragments hundreds of base pairs longThe 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).

11. 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]

12. 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]

13. Principle of DHPLC  Rapid denaturation of DNA by heating, re-annealing by slow cooling.  Heteroduplexes form in the presence of two different alleles.

14. DHPLC Graphical Scheme

15. Software Software 1. HSM (Hitachi) and WAVEMAKER 4.1 (Transgenomic) software 2. Navigator (Transgenomic).

18. Nucleic Acid Amplifications e.g. PCR  PCR Amplification for Mutation Analysis Using the Transgenomic WAVE® System from Invitrogen Discoverase™ dHPLC DNA Polymerase a proofreading ability by virtue of its 3’ to 5’ exonuclease activity increases fidelity an optimized buffer that improves enzyme fidelity –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. Transgenomic WAVE® System –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.

19. 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.

20. 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, Sigma- Aldrich). 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).

21. HomoduplexesHeteroduplexes 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.

22. 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.

23. Applications  DHPLC Systems (e.g. WAVE Systems) can be used for a wide range of applications including: –Mutation Detection –Sizing –Oligonucleotide Purification and Analysis –Forensics –Microbial Analysis –RNA Isolation and Purification –Single Base Extension –Methylation

24. 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/Primer- based Amplification Technologies

25. 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

26. CARTRIDGE dependent Applications e.g. via WAVE Systems Sizing and Mutation Detection  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 especially for Rapid Mutation Detection  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 for Oligo Purification work only  NUC-99-3550 OligoSep® Cartridge 4.6 mm x 50 mm; for Oligo Purification work only for Oligo Purification  NUC-99-3860 OligoSep Prep HC Cartridge 7.8 mm x 50 mm, for Oligo Purification, load capacity of 5000µg for variety of RNA applications  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

30. 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.

31. Example of Oligo Analysis by DHPLC

33. Thanks for Your Attentions Reference:

34. 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.