Noncontinuously Binding Loop-Out Primers for Avoiding Problematic DNA Sequences in PCR and Sanger Sequencing  Kelli Sumner, Jeffrey J. Swensen, Melinda.

Slides:



Advertisements
Similar presentations
Measurement of Relative Copy Number of CDKN2A/ARF and CDKN2B in Bladder Cancer by Real-Time Quantitative PCR and Multiplex Ligation-Dependent Probe Amplification.
Advertisements

Nine Novel Germline Gene Variants in the RET Proto-Oncogene Identified in Twelve Unrelated Cases Syed A. Ahmed, Karen Snow-Bailey, W. Edward Highsmith,
CyclinD1/CyclinD3 Ratio by Real-Time PCR Improves Specificity for the Diagnosis of Mantle Cell Lymphoma Carol D. Jones, Katherine H. Darnell, Roger A.
Quantification of Parvovirus B19 DNA Using COBAS AmpliPrep Automated Sample Preparation and LightCycler Real-Time PCR Stefan Schorling, Gunnar Schalasta,
Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis  Philippe Szankasi, David.
Keyur P. Patel, Bedia A. Barkoh, Zhao Chen, Deqin Ma, Neelima Reddy, L
Triplet Repeat Primed PCR Simplifies Testing for Huntington Disease
Detection of Exon 12 Mutations in the JAK2 Gene
Development of a Next-Generation Sequencing Method for BRCA Mutation Screening  Maurice Chan, Shen Mo Ji, Zhen Xuan Yeo, Linda Gan, Eric Yap, Yoon Sim.
A Multiplex SNaPshot Assay as a Rapid Method for Detecting KRAS and BRAF Mutations in Advanced Colorectal Cancers  Sandrine Magnin, Erika Viel, Alice.
Establishment and Study of Different Real-Time Polymerase Chain Reaction Assays for the Quantification of Cells with Deletions of Chromosome 7  Elia Mattarucchi,
Triplet Repeat Primed PCR Simplifies Testing for Huntington Disease
Michael T. Seipp, Jacob D. Durtschi, Michael A
Increased Sensitivity of the Roche COBAS AMPLICOR HCV Test, Version 2
Philippe Szankasi, Mohamed Jama, David W. Bahler 
Comparison of High-Resolution Melting Analysis, TaqMan Allelic Discrimination Assay, and Sanger Sequencing for Clopidogrel Efficacy Genotyping in Routine.
Molecular Diagnosis of Autosomal Dominant Polycystic Kidney Disease Using Next- Generation Sequencing  Adrian Y. Tan, Alber Michaeel, Genyan Liu, Olivier.
Silke Lassmann, Ulrike V
Cornelis J. J. Huijsmans, Jeroen Poodt, Paul H. M. Savelkoul, Mirjam H
Hou-Sung Jung, Gregory J. Tsongalis, Joel A. Lefferts 
A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2  Jianli Li, Hongzheng Dai, Yanming Feng, Jia Tang, Stella Chen,
Long-Range (17.7 kb) Allele-Specific Polymerase Chain Reaction Method for Direct Haplotyping of R117H and IVS-8 Mutations of the Cystic Fibrosis Transmembrane.
Molecular Typing of West Nile Virus, Dengue, and St
Detecting 22q11.2 Deletions by Use of Multiplex Ligation-Dependent Probe Amplification on DNA from Neonatal Dried Blood Spot Samples  Karina M. Sørensen,
Getting Things Backwards to Prevent Primer Dimers
Patrick R. Murray  The Journal of Molecular Diagnostics 
Clinical and Analytical Sensitivities in Hereditary Hemorrhagic Telangiectasia Testing and a Report of de Novo Mutations  Friederike Gedge, Jamie McDonald,
Detection of an Apparent Homozygous 3120G>A Cystic Fibrosis Mutation on a Routine Carrier Screen  Denise LaMarche Heaney, Patrick Flume, Lauren Hamilton,
Andrea Gaedigk, Amanda K. Riffel, J. Steven Leeder 
Christine L. Baker, Cecily P. Vaughn, Wade S. Samowitz 
Verification of Wild-Type EGFR Status in Non–Small Cell Lung Carcinomas Using a Mutant-Enriched PCR on Selected Cases  Yi-Lin Chen, Cheng-Chan Lu, Shu-Ching.
SeqSharp The Journal of Molecular Diagnostics
Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis  Philippe Szankasi, David.
Detection of Exon 12 Mutations in the JAK2 Gene
Development and Clinical Implementation of a Combination Deletion PCR and Multiplex Ligation-Dependent Probe Amplification Assay for Detecting Deletions.
Comprehensive Diagnostic Testing for Stereocilin
Catherine E. Keegan, Anthony A. Killeen 
Development of a Next-Generation Sequencing Method for BRCA Mutation Screening  Maurice Chan, Shen Mo Ji, Zhen Xuan Yeo, Linda Gan, Eric Yap, Yoon Sim.
Multiplex Amplification Coupled with COLD-PCR and High Resolution Melting Enables Identification of Low-Abundance Mutations in Cancer Samples with Low.
Keyur P. Patel, Bedia A. Barkoh, Zhao Chen, Deqin Ma, Neelima Reddy, L
Utility of NIST Whole-Genome Reference Materials for the Technical Validation of a Multigene Next-Generation Sequencing Test  Bennett O.V. Shum, Ilya.
Benjamin P. Song, Surbhi Jain, Selena Y. Lin, Quan Chen, Timothy M
BRAF Mutation Testing in Solid Tumors
A Rare Mutation in the Primer Binding Region of the Amelogenin Gene Can Interfere with Gender Identification  Bonnie Shadrach, Mairead Commane, Carol.
Genotyping Single Nucleotide Polymorphisms in Human Genomic DNA with an Automated and Self-Contained PCR Cassette  Dammika P. Manage, Lucy Ma, Jana Lauzon,
A Multi-Exonic BRCA1 Deletion Identified in Multiple Families through Single Nucleotide Polymorphism Haplotype Pair Analysis and Gene Amplification with.
Larissa V. Furtado, Helmut C. Weigelin, Kojo S. J
Multiplex Amplification Coupled with COLD-PCR and High Resolution Melting Enables Identification of Low-Abundance Mutations in Cancer Samples with Low.
Larissa V. Furtado, Helmut C. Weigelin, Kojo S. J
An Allele-Specific PCR System for Rapid Detection and Discrimination of the CYP2C19∗4A, ∗4B, and ∗17 Alleles  Stuart A. Scott, Qian Tan, Usman Baber,
Evaluating the Effect of Unclassified Variants Identified in MMR Genes Using Phenotypic Features, Bioinformatics Prediction, and RNA Assays  Lucia Pérez-Cabornero,
A Novel Long-Range PCR Sequencing Method for Genetic Analysis of the Entire PKD1 Gene  Ying-Cai Tan, Alber Michaeel, Jon Blumenfeld, Stephanie Donahue,
Ye Bang-Ce, Chu Xiaohe, Fan Ye, Li Songyang, Yin Bincheng, Zuo Peng 
Retrospective Comparison of Nucleic Acid Sequence–Based Amplification, Real-Time PCR, and Galactomannan Test for Diagnosis of Invasive Aspergillosis 
Next-Generation Sequencing for Infectious Disease Diagnosis and Management  Martina I. Lefterova, Carlos J. Suarez, Niaz Banaei, Benjamin A. Pinsky  The.
Simultaneous Amplification, Detection, and Analysis of Common Mutations in the Galactose-1-Phosphate Uridyl Transferase Gene  Mohamed Jama, Lesa Nelson,
Noncontinuously Binding Loop-Out Primers for Avoiding Problematic DNA Sequences in PCR and Sanger Sequencing  Kelli Sumner, Jeffrey J. Swensen, Melinda.
Multiple Sequence Variants in Hereditary Hemorrhagic Telangiectasia Cases  Jamie McDonald, Friederike Gedge, Allene Burdette, James Carlisle, Changkuoth.
Avoiding Pitfalls in Molecular Genetic Testing
Amplification Refractory Mutation System, a Highly Sensitive and Simple Polymerase Chain Reaction Assay, for the Detection of JAK2 V617F Mutation in Chronic.
Processed Pseudogene Confounding Deletion/Duplication Assays for SMAD4
Feras M. Hantash, Arlene Rebuyon, Mei Peng, Joy B
Danielle C. Smith, Alina Esterhuizen, Jacquie Greenberg 
Cecily P. Vaughn, Elaine Lyon, Wade S. Samowitz 
Novel Method for PIK3CA Mutation Analysis
Nathan D. Montgomery, Sara R. Selitsky, Nirali M. Patel, D
Characterization of a Recurrent Novel Large Duplication in the Cystic Fibrosis Transmembrane Conductance Regulator Gene  Feras M. Hantash, Joy B. Redman,
Measurement of Relative Copy Number of CDKN2A/ARF and CDKN2B in Bladder Cancer by Real-Time Quantitative PCR and Multiplex Ligation-Dependent Probe Amplification 
The History and Impact of Molecular Coding Changes on Coverage and Reimbursement of Molecular Diagnostic Tests  Susan J. Hsiao, Mahesh M. Mansukhani,
Mitochondrial DNA as a Cancer Biomarker
Presentation transcript:

Noncontinuously Binding Loop-Out Primers for Avoiding Problematic DNA Sequences in PCR and Sanger Sequencing  Kelli Sumner, Jeffrey J. Swensen, Melinda Procter, Mohamed Jama, Whitney Wooderchak-Donahue, Tracey Lewis, Michael Fong, Lindsey Hubley, Monica Schwarz, Youna Ha, Eleri Paul, Benjamin Brulotte, Elaine Lyon, Pinar Bayrak-Toydemir, Rong Mao, Genevieve Pont-Kingdon, D. Hunter Best  The Journal of Molecular Diagnostics  Volume 16, Issue 5, Pages 477-480 (September 2014) DOI: 10.1016/j.jmoldx.2014.04.005 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Sample containing the MEN1 rs509606 polymorphism (not shown); sequences from c.240_259. A: Amplification with a normal forward primer; the 4-bp deletion (black bar), c.249_252delGTCT, was undetectable. B: Amplification with a loop-out forward primer showed a clearly discernable 4-bp deletion, c.249_252delGTCT. The Journal of Molecular Diagnostics 2014 16, 477-480DOI: (10.1016/j.jmoldx.2014.04.005) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Amplification of ASS1 exon 9. Top: Amplification through the problematic region with a traditional primer. The sequencing signal decreased to zero shortly after the G-rich region. Bottom: Amplification with a loop-out forward primer removed the problematic region, resulting in consistent sequence signal throughout the read. The Journal of Molecular Diagnostics 2014 16, 477-480DOI: (10.1016/j.jmoldx.2014.04.005) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions