Name: wild-type blocking pcr combined with direct sequencing as a highly sensitive method for detection of low-frequency somatic mutations
Uploaded: 2017-03-29T15:00:00
Description: Watch this Scientific Journal Video about Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations at JoVE.com

The authors have no acknowledgements.

Ethics Statement: All testing of human samples was performed after obtaining Institutional Review Board (IRB) approval.
1. DNA Extraction from FFPE Tissue, Peripheral Blood, and Bone Marrow Aspirate
<ol>
	<li>For bone marrow FFPE tissue with DNA FFPE extraction kit
	<ol>
		<li>Begin with FFPE tissue from unstained slides (5 - 10 sections at 5 - 10 &#181;m thickness). 
		NOTE: If beginning with tissue shavings, use 3 - 6 sections at 5 - 10 &#181;m thickness and skip to step 1.1.6.</li>
	...

<ol>
	<li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR.">Digital PCR.</a> Proc Natl Acad of Sci U S A. 96 (16), 9236-9241 (1999).</li><li>Milbury, C. A., Li, J., Makrigiorgos, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR-based+methods+for+the+enrichment+of+minority+alleles+and+mutations.">PCR-based methods for the enrichment of minority alleles and mutations.</a> Clin Chem. 55 (4), 632-640 (2009).</li><li>Albitar, A., Ma, W., DeDios, I., Estella, J., Agersborg, S., Albitar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+and+high+sensitivity+test+for+MYD88+mutations+using+locked+nucleic+acid.">Positive selection and high sensitivity test for MYD88 mutations using locked nucleic acid.</a> Int J Lab Hematol. 38 (2), 133-140 (2016).</li><li>Dominguez, P. L., Kolodney, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild-type+blocking+polymerase+chain+reaction+for+detection+of+single+nucleotide+minority+mutations+from+clinical+specimens.">Wild-type blocking polymerase chain reaction for detection of single nucleotide minority mutations from clinical specimens.</a> Oncogene. 24 (45), 6830-6834 (2005).</li><li>Gray, P. N., Dunlop, C. L. M., Elliott, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+All+Next+Generation+Sequencing+Diagnostics+are+Created+Equal:+Understanding+the+Nuances+of+Solid+Tumor+Assay+Design+for+Somatic+Mutation+Detection.">Not All Next Generation Sequencing Diagnostics are Created Equal: Understanding the Nuances of Solid Tumor Assay Design for Somatic Mutation Detection.</a> Cancers. 7 (3), 1313-1332 (2015).</li><li>Smith, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biased+estimates+of+clonal+evolution+and+subclonal+heterogeneity+can+arise+from+PCR+duplicates+in+deep+sequencing+experiments.">Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments.</a> Genome Biol. 15 (8), 420 (2014).</li><li>Varettoni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+clinical+significance+of+the+MYD88+(L265P)+somatic+mutation+in+Waldenstr&#246;m's+macroglobulinemia+and+related+lymphoid+neoplasms.">Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstr&#246;m's macroglobulinemia and related lymphoid neoplasms.</a> Blood. 121 (13), 2522-2528 (2013).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MYD88+L265P+in+Waldenstr&#246;m+macroglobulinemia,+immunoglobulin+M+monoclonal+gammopathy,+and+other+B-cell+lymphoproliferative+disorders+using+conventional+and+quantitative+allele-specific+polymerase+chain+reaction.">MYD88 L265P in Waldenstr&#246;m macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction.</a> Blood. 121 (11), 2051-2058 (2013).</li><li>Gertz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waldenstr&#246;m+macroglobulinemia:+2012+update+on+diagnosis,+risk+stratification,+and+management.">Waldenstr&#246;m macroglobulinemia: 2012 update on diagnosis, risk stratification, and management.</a> Amer J Hematol. 87 (5), 503-510 (2012).</li><li>Salar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MYD88+(L265P)+Mutation+Confers+Very+Poor+Response+and+Outcome+after+Second-Line+Therapy+in+Patients+with+Diffuse+Large+B-Cell+Lymphoma+(DLBCL).">MYD88 (L265P) Mutation Confers Very Poor Response and Outcome after Second-Line Therapy in Patients with Diffuse Large B-Cell Lymphoma (DLBCL).</a> Blood. 124 (21), 1690-1690 (2014).</li><li>Ngo, V. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenically+active+MYD88+mutations+in+human+lymphoma.">Oncogenically active MYD88 mutations in human lymphoma.</a> Nature. 470 (7332), 115-119 (2011).</li><li>Pasqualucci, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+coding+genome+of+diffuse+large+B-cell+lymphoma.">Analysis of the coding genome of diffuse large B-cell lymphoma.</a> Nat Genet. 43 (9), 830-837 (2011).</li><li>Dieffenbach, C., Lowe, T., Dveksler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+concepts+for+PCR+primer+design.">General concepts for PCR primer design.</a> PCR Methods Appl. 3 (3), S30-S37 (1993).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Applied Biosystems. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> User Guide for Applied Biosystems 3730/3730xl DNA Analyzer. , (2014).</li><li>Applied Biosystems. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> User Guide for SeqScape Software 3. , (2012).</li><li>Mouritzen, P., Nielsen, A. T., Pfundheller, H. M., Choleva, Y., Kongsbak, L., M&#248;ller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphism+genotyping+using+locked+nucleic+acid+(LNA).">Single nucleotide polymorphism genotyping using locked nucleic acid (LNA).</a> Expert Rev Mol Diagn. 3 (1), 27-38 (2003).</li><li>Quach, N., Goodman, M. F., Shibata, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+mutation+artifacts+after+formalin+fixation+and+error+prone+translesion+synthesis+during+PCR.">In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR.</a> BMC Clin Pathol. 4 (1), (2004).</li><li>Gallegos Ruiz, M. I., Floor, K., Rijmen, F., Gr&#252;nberg, K., Rodriguez, J. A., Giaccone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+and+K-ras+mutation+analysis+in+non-small+cell+lung+cancer:+comparison+of+paraffin+embedded+versus+frozen+specimens.">EGFR and K-ras mutation analysis in non-small cell lung cancer: comparison of paraffin embedded versus frozen specimens.</a> Anal Cell Pathol. 29 (3), 257-264 (2007).</li><li>Solassol, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KRAS+mutation+detection+in+paired+frozen+and+formalin-fixed+paraffin-embedded+(FFPE)+colorectal+cancer+tissues.">KRAS mutation detection in paired frozen and formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues.</a> Int J Mol Sci. 12 (5), 3191-3204 (2011).</li><li>Yost, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+high-confidence+somatic+mutations+in+whole+genome+sequence+of+formalin-fixed+breast+cancer+specimens.">Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens.</a> Nucleic Acids Res. 40 (14), e107-e107 (2012).</li><li>Do, H., Wong, S. Q., Li, J., Dobrovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+sequence+artifacts+in+amplicon-based+massively+parallel+sequencing+of+formalin-fixed+paraffin-embedded+DNA+by+enzymatic+depletion+of+uracil-containing+templates.">Reducing sequence artifacts in amplicon-based massively parallel sequencing of formalin-fixed paraffin-embedded DNA by enzymatic depletion of uracil-containing templates.</a> Clin Chem. 59 (9), 1376-1383 (2013).</li><li>Oldenburg, R. P., Liu, M. S., Kolodney, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+amplification+of+rare+mutations+using+locked+nucleic+acid+oligonucleotides+that+competitively+inhibit+primer+binding+to+wild-type+DNA.">Selective amplification of rare mutations using locked nucleic acid oligonucleotides that competitively inhibit primer binding to wild-type DNA.</a> J Invest Dermatol. 128 (2), 398-402 (2008).</li><li>Mancini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+novel+methods+for+rapid+detection+and+quantification+of+DNMT3A+R882+mutations+in+acute+myeloid+leukemia.">Two novel methods for rapid detection and quantification of DNMT3A R882 mutations in acute myeloid leukemia.</a> J Mol Diagn. 17 (2), 179-184 (2015).</li><li>Parsons, B. L., Heflich, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+selection+methods+for+the+direct+analysis+of+point+mutations.">Genotypic selection methods for the direct analysis of point mutations.</a> Mutat Res Rev Muta Res. 387 (2), 97-121 (1997).</li><li>Liu, Q., Swiderski, P., Sommer, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Truncated+amplification:+a+method+for+high-fidelity+template-driven+nucleic+acid+amplification.">Truncated amplification: a method for high-fidelity template-driven nucleic acid amplification.</a> BioTechniques. 33 (1), 129-139 (2002).</li><li>Kaur, M., Zhang, Y., Liu, W. H., Tetradis, S., Price, B. D., Makrigiorgos, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligation+of+a+primer+at+a+mutation:+a+method+to+detect+low+level+mutations+in+DNA.">Ligation of a primer at a mutation: a method to detect low level mutations in DNA.</a> Mutagenesis. 17 (5), 365-374 (2002).</li><li>Albitar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Sensitivity+Testing+Shows+Multiclonal+Mutations+in+Patients+with+CLL+Treated+with+BTK+Inhibitor+and+Lack+of+Mutations+in+Ibrutinib-Naive+Patients.">High Sensitivity Testing Shows Multiclonal Mutations in Patients with CLL Treated with BTK Inhibitor and Lack of Mutations in Ibrutinib-Naive Patients.</a> Blood. 126 (23), 716 (2015).</li></ol>

The WTB-PCR assay described here uses a generic set of primers with a blocking oligo designed to block amplification of WT DNA during extension (Figure 1). The WTB-PCR product is then sequenced for mutational analysis. The utility of WTB-PCR/Sanger lies in its simplicity, high-sensitivity, and high-throughput. Using the guidelines described here, most existing Sanger based assays can be simply modified via the addition of a blocking oligonucleotide to greatly increase sensitivity. In the example assay pr...

Sanger sequencing has traditionally been the gold standard in testing for both known and unknown somatic mutations. One of the limitations of Sanger sequencing is its limit of detection (~ 10 - 20% mutant allele in a background of WT)1. This level of sensitivity is inappropriate for detecting low level somatic mutations that may be present in samples from premalignant tissues or patients with few circulating tumor cells, or when bone marrow (BM) is patchy. This also makes assessing residual disease after therapy or detecting emerging resistance mutations during therapy difficult by conventional sequencing alone2. By replacing conventional PCR with locked nucleic acid (LNA)-mediated wild-type blocking PCR (WTB-PCR) in Sanger sequencing, sensitivities of up to 0.1% mutant allele in a background of WT can be achieved2,3,4. In WTB-PCR, enrichment for mutant alleles is achieved via the addition of a short (~ 10 - 14 NT) blocking (LNA) oligonucleotide that binds preferentially to WT DNA thereby preventing amplification of WT DNA. The mutant enriched WTB-PCR product can then be sequenced. By blocking WT DNA rather than selecting for specific mutations WTB-PCR allows for enrichment of both known and unknown mutations present in minute cell fractions.
Multiple methods are currently used for detecting mutations in small cells fractions. This includes allele-specific PCR, amplification-refractory mutation system (ARMS), denaturing high performance liquid chromatography (DHPLC), beads, emulsions, amplification, and magnetics (BEAMing), electric field-induced release and measurement (EFIRM), high resolution melting point and others. However, most of these methods are limited by false-positives and the ability to only detect one mutation that the assay was designed for4. WTB-PCR, however, allows the user to visualize sequencing traces which enables the detection of multiple mutation types and can aid in ruling out false positives due to artifacts or deamination events. Next-generation sequencing (NGS) may offer a suitable alternative to conventional sequencing, however, substantially greater costs, complexity, and longer assay time render it an unnecessary option for many disease types with few distinct molecular markers or for monitoring patients on therapy for emerging resistance mutations. Furthermore, high false positive rates when detecting variants with mutant allele frequencies of less than 5% can pose a problem for amplicon-based NGS5,6.
Here we demonstrate the increase in sensitivity achieved by WTB-PCR in screening for mutations in the myeloid differentiation factor 88 gene as described by Albitar et al.3 MYD88 mutations are important diagnostic and prognostic factors in Waldenstr&#246;m&#39;s Macroglobulinemia (WM), IgM monoclonal gammopathy of unknown significance (IgM-MGUS), splenic marginal zone lymphoma (SMZL), and diffuse large B-cell lymphoma (DLBCL). MYD88 mutations are found in almost all cases of WM and approximately 50% of patients with Immunoglobulin M (IgM)-secreting MGUS. Contrastingly, MYD88 mutations are found in only 0-6% of patients with SMZL and are absent in multiple myeloma7,8. Because overlapping morphologic, immunophenotypic, cytogenetic, and clinical characteristics between WM and SMZL or IgM-multiple myeloma can often complicate differential diagnoses, the presence of a MYD88 mutation may serve as a useful identifying factor9. MYD88 mutations have also been associated with greater disease burden in patients with DLBCL and poor overall survival following therapy7,10. Additionally, because MYD88 mutations are more frequently found in activated B-cell-like (ABC) DLBCL than germinal center B-cell-like (GCB) DLBCL or primary mediastinal B-cell lymphoma (PMBL), MYD88 mutation status may serve as a surrogate marker for the ABC subtype11,12.
The detailed protocol provided here serves as a template from which new assays can be developed or most existing sequencing assays can be easily adapted to accurately detect low frequency mutations in various sample types. The approach can also be used for monitoring and detecting resistant mutations that may develop in tumors or even bacteria that may develop while patients are being treated with targeted therapy or antibiotics. Furthermore, it addresses and remedies many of the issues associated with mutation enrichment, particularly in formalin-fixed paraffin-embedded (FFPE) tissue.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 or 2 ml Safe-Lock microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>05-402-25</td>
			<td></td>
		</tr>
		<tr>
			<td>100% alcohol</td>
			<td>VWR</td>
			<td>89370-084</td>
			<td>Histology grade; 91.5% Ethanol, 5% Isopropyl alcohol, 4.5% Methyl alcohol</td>
		</tr>
		<tr>
			<td>3730XL sequencer</td>
			<td>ABI</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>For magnetic bead PCR purification</td>
		</tr>
		<tr>
			<td>Aluminum sealing foils</td>
			<td>GeneMate</td>
			<td>T-2451-1</td>
			<td>For PCR and cold storage</td>
		</tr>
		<tr>
			<td>BigDye Terminator v3.1 Cycle sequencing kit</td>
			<td>Life Technologies</td>
			<td>4337455</td>
			<td>For bi-directional sequencing. With 5X Sequencing Buffer</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 Series</td>
			<td>Eppendorf</td>
			<td></td>
			<td>A-2-DWP rotor (for PCR plate)</td>
		</tr>
		<tr>
			<td>Cold plate for 96 well plates</td>
			<td>Eppendorf</td>
			<td>Z606634</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse, RNAse-free, ultra-pure water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (100mM)</td>
			<td>Invitrogen</td>
			<td>10297-117</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-96 Side-Skirted Magnet</td>
			<td>Thermo Fisher Scientific</td>
			<td>12027</td>
			<td>For use in PCR Purification.</td>
		</tr>
		<tr>
			<td>Ethanol Absolute&#160;</td>
			<td>Sigma</td>
			<td>E7023</td>
			<td>200 proof, for molecular biology</td>
		</tr>
		<tr>
			<td>Exiqon website Oligo Tools</td>
			<td></td>
			<td></td>
			<td><a href="http://www.exiqon.com/oligo-tools">www.exiqon.com/oligo-tools</a></td>
		</tr>
		<tr>
			<td>FastStart Taq DNA polymerase (5 U/ul)</td>
			<td>Roche</td>
			<td>12032937001</td>
			<td>With10X concentrated PCR reaction buffer, with 20 mM MgCl2&#160;</td>
		</tr>
		<tr>
			<td>Gel electrophoresis apparatus</td>
			<td></td>
			<td></td>
			<td>2% agarose gel</td>
		</tr>
		<tr>
			<td>GeneRead DNA FFPE extraction Kit&#160;</td>
			<td>Qiagen</td>
			<td>180134</td>
			<td>Contains uracil DNA glycosylase necessary for reducing sequencing artifacts</td>
		</tr>
		<tr>
			<td>Hi-Di Formamide</td>
			<td>ABI</td>
			<td>4311320</td>
			<td>For sequencing.</td>
		</tr>
		<tr>
			<td>LNA oligonucleotide</td>
			<td>Exiqon</td>
			<td>500100</td>
			<td>5&#39;-TCAGA+AG+C+G+A+C+T+G+A+T+CC/invdT/ (+N = LNA bases)</td>
		</tr>
		<tr>
			<td>M13-F Sequencing Primer</td>
			<td>ABI</td>
			<td></td>
			<td>5&#39;-tgt aaa acg acg gcc agt</td>
		</tr>
		<tr>
			<td>M13-R Sequencing Primer</td>
			<td>ABI</td>
			<td></td>
			<td>5&#39;-cag gaa aca gct atg acc</td>
		</tr>
		<tr>
			<td>Mastercycler Pro S Thermocycler</td>
			<td>Eppendorf</td>
			<td>E950030020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Model 5430</td>
			<td>Eppendorf</td>
			<td></td>
			<td>FA-45-30-11 rotor (for 1.5/2 ml microcentrifuge tubes)</td>
		</tr>
		<tr>
			<td>NanoDrop 2000 Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR forward primer</td>
			<td>IDT</td>
			<td></td>
			<td>5&#39;-tgt aaa acg acg gcc agt TGC CAG GGG TAC TTA GAT GG</td>
		</tr>
		<tr>
			<td>PCR reverse primer</td>
			<td>IDT</td>
			<td></td>
			<td>5&#39;-cag gaa aca gct atg acc GGT TGG TGT AGT CGC AGA CA</td>
		</tr>
		<tr>
			<td>PCR plates</td>
			<td>GeneMate</td>
			<td>T-3107-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors</td>
			<td></td>
			<td></td>
			<td>20, 200, 1000 &#181;l</td>
		</tr>
		<tr>
			<td>Plate septa, 96 well</td>
			<td>ABI</td>
			<td>4315933</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>Qiagen</td>
			<td>51304</td>
			<td>For BM aspirate and peripheral blood</td>
		</tr>
		<tr>
			<td>SeqScape Sortware v3.0</td>
			<td>ABI</td>
			<td>4474978</td>
			<td>For sequencing analysis</td>
		</tr>
		<tr>
			<td>Slide basket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate (3M, pH 5.2)&#160;</td>
			<td>Sigma</td>
			<td>S7899</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile filtered pipette tips&#160;</td>
			<td></td>
			<td></td>
			<td>20, 200, 1000 &#181;l</td>
		</tr>
		<tr>
			<td>Thermomixer C&#160;</td>
			<td>Eppendorf</td>
			<td>5382000023</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex genie</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash reservoir</td>
			<td></td>
			<td></td>
			<td>~1000 ml</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>89370-088</td>
			<td>Histology grade</td>
		</tr>
	</tbody>
</table>

wild-type blocking pcr combined with direct sequencing as a highly sensitive method for detection of low-frequency somatic mutations

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.

A conceptual overview of WTB-PCR during extension is presented in Figure 1. Because a single nucleotide mismatch in the blocker-DNA hybrid greatly decreases its melting temperature (&#916;Tm=20 - 30 &#176;C), amplification of the WT allele is blocked while mutant template DNA is free to complete extension17. In this manner, mutant DNA is amplified exponentially while WT DNA is amplified linearly.
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Watch this Scientific Journal Video about Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations at JoVE.com

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

The overall goal of this procedure is to detect low frequency somatic mutations in a variety of sample types. This methodology can help answer key questions in somatic mutation testing by facilitating the detection of very low frequency mutations. Here we will look for mutations in the myd88 gene in bone marrow samples.The main advantage of this technique is that it provides highly accurate and sensitive information about the presence of somatic mutations, even with very few neoplastic cells. This wild type blocking PCR based sequencing assay was developed to amplify part of exon five in the myd88 gene, ensuring coverage of the L265 hotspot. The forward and reverse primers were designed with a five prime M13 sequence to allow for a kneeling of complimentary sequencing primers.Design the blocking oligonucleotide to be approximately 10 to 15 bases in length and complimentary to the wild type template where mutant enrichment is desired. A shorter oligo will improve mismatch discrimination. To achieve high target specificity it's important not to use too much of the blocking nucleotides, as this will result in very sticky oligonucleotides.To design the blocking oligonucleotide begin by navigating to the Oligo Tools website. Select the Oligo TM prediction tool. A new window will open up.Paste the sequence of the wild type template to be blocked into the oligo sequence box. Add a plus sign in front of the blocker bases to mark them. Click on the calculate button to determine the approximate TM of the DNA blocker hybrid.The calculated melting temperatures will appear in the boxes below. Design the blocking oligo to have a melting temperature 10 to 15 degrees Celsius above the extension temperature during thermocycling. Here, the extension temperature is 72 degrees Celsius.To adjust the melting temperature, add, remove, or substitute blocking bases. Avoid long stretches of three to four blocking C or G bases. Next, to avoid secondary structure formation or self dimerization go back to the Oligo Tools website home screen and select the Oligo Optimizer tool.A new window will open. Paste the sequence of the wild type template to be blocked into the box. Add a plus sign to indicate blocking bases.Select the two boxes for secondary structure and self only and press the analyze button to see the scores for hybridization and secondary structure. These scores represent very rough estimates of the melting temperatures of the self dimers and secondary structures respectively. Lower scores are optimal and can be achieved by limiting blocker blocker pairing.Remove or reposition blocking nucleotides in order to achieve lower scores. The optimized blocking oligo nucleotide for myd88 shown here strikes a balance between the DNA blocker hybrid melting temperature and low enough hybridization and secondary structure scores. It was designed to cover amino acids Q262 to I266, and features a three prime inverted DT to inhibit both extension by DNA polymerase and degradation by three prime exonuclease.Once the primers have been designed set up the wild type blocking PCR and perform thermocycling as described in the accompanying document. Remove magnetic beads from storage at four degrees Celsius and bring them to room temperature. Transfer 10 microliters of the PCR product to a new PCR plate.Vortex the magnetic beads vigorously to fully resuspend the magnetic particles and then add 18 microliters of magnetic beads to each well on the new plate. Pipette up and down 10 times to mix. Then incubate the plate at room temperature for five minutes.Following the incubation, place the PCR plate onto the side skirted magnet plate for two minutes to separate the beads from the solution. Use a multi channel pipette to aspirate the supernatant. Take care to avoid the bead pellet.Next, dispense 150 microliters of 70%ethanol into each well and incubate the plate at room temperature for at least 30 seconds. Then aspirate the ethanol with a multichannel pipette and discard the tips. Repeat this wash procedure once more.Using a 20 microliter multichannel pipette aspirate the remaining ethanol from each well and discard the tips. After allowing about 10 minutes for the wells of the plate to dry remove it from the magnet and add 40 microliters of nuclease free water to each well. Pipette up and down 15 times to mix.Then incubate at room temperature for two minutes. After incubating, place the PCR plate back onto the magnet plate for one minute to separate the beads from solution. Transfer 35 microliters of purified product to a new PCR plate, and perform bidirectional sequencing as described in the accompanying document.Once bidirectional sequencing has been performed to purify the sequencing products prepare a fresh one in 25 solution of three molar sodium acetate at PH 5.2 and 100%ethanol. Also prepare a fresh solution of 70%ethanol. To each well of both the forward and reverse sequencing plates add 30 microliters of sodium acetate in 100%ethanol and pipette up and down five times to mix.Then reseal the plate, and incubate in the dark at room temperature for 20 minutes. After 20 minutes have passed, centrifuge the plate at 2, 250 times G for 15 minutes. After the spin, remove the plate sealer, and invert the plate once over a waste container.If the plate is inverted multiple times the pellets may loosen from the well bottoms. Place the inverted plate on a clean paper towel, and centrifuge at 150 times G for one minute. Next, add 150 microliters of 70%ethanol to each well and reseal the plate.Spin at 2, 250 times G for five minutes. Then repeat the process of removing the plate sealer and inverting the plate. If the wells are not completely dry, allow them to air dry at room temperature.Make sure the samples are protected from light. Once the wells are completely dry add 10 microliters of formamide to each well and pipette up and down 10 times to mix. Reseal the plate.Denature using a thermo cycler at 95 degrees Celsius for three minutes followed by four degrees Celsius for five minutes. After denaturing, replace the plate sealer with a septa and sequence on a sequencing platform according to the manufacturer's instructions. Using sequence analysis software, visualize the traces, and align the sequences to the appropriate reference sequences.Align myd88 to NCBI reference sequence NM002468. Genomic DNA from patients with and without mutations underwent both conventional and wild type blocking PCR and the resulting PCR products were then sequenced. As can be seen here, the mutant allele was enriched when wild type blocking PCR was performed, and false positives were not seen in wild type DNA.A characteristic drop off in signal intensity as shown here is often seen if too high a concentration of blocker is used, or if post PCR purification failed to remove blocker prior to bidirectional sequencing. This occurs when enzymatic purification is performed in place of magnetic bead purification. Any PCR based assay that enriches for mutant alleles will detect low frequency artifacts.These traces show an increase in CG sequencing artifacts, relative to TA artifacts in FFPE tissue when cytosine or methylated cytosine are deaminated via formal infixation to uracil or thiamine respectively. Uracil DNA glycosylase or UDG can excise uracil prior to wild type blocking PCR, helping to reduce sequencing artifacts. However, thiamine resulting from deaminated five methyl cytosine which frequently occurs at CPG islands cannot be excised by UDG.Decreasing the concentration of blocker used in wild type blocking PCR may help to reduce the occurrence of sequencing artifacts that are not remedied by UDG treatment. After watching this video, you should have a good understanding of how to test somatic mutations with a high degree of accuracy and sensitivity, using the wild type blocking technique. The principle of this technology can be applied to the detection of mutations in small sub populations of cells.Therefore, it has utility in detecting minimal residual disease, monitoring patients, and predicting early relapse in patients with various neoplasms.

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