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.

Etikk Uttalelse: All testing av humane prøver ble utført etter å skaffe Institutional Review Board (IRB) godkjennelse. 1. DNA-ekstraksjon fra FFPE Tissue, perifert blod, og benmargsaspirat <ol><li> For benmarg FFPE vevet med DNA FFPE ekstraksjonssett <ol><li> Begynn med FFPE vev fra ufargede bakker (5 - 10 seksjoner ved 5 - 10 um tykkelse). MERK: Hvis starten med vev spon, bruke 3 - 6 deler ved 5 - 10 um tykkelse og går videre til trinn 1.1.6. </li><li> Plasser glir i en glide k...

<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>

WTB-PCR-assay som er beskrevet her anvender en generisk sett av primere med en blokkerende oligo utformet for å blokkere amplifikasjon av WT-DNA under forlengelse (figur 1). WTB-PCR-produktet blir deretter sekvensert for mutasjonsanalyse. Anvendeligheten av WTB-PCR / Sanger ligger i dens enkelhet, høy følsomhet og høy gjennomstrømming. Ved å bruke de retningslinjer som er beskrevet her, kan de fleste eksisterende Sanger-baserte analyser på enkel måte modifiseres ved tilsetning av et blokkerings ...

Sanger-sekvensering har tradisjonelt vært gullstandarden i testing for både kjente og ukjente somatiske mutasjoner. En av begrensningene ved Sanger-sekvensering er deteksjonsgrensen (~ 10 - 20% mutant allel i en bakgrunn av WT) 1. Dette nivået av følsomhet er upassende for å detektere lavt nivå somatiske mutasjoner som kan være tilstede i prøver fra pre-maligne vev eller pasienter med noen sirkulerende tumorceller, eller når benmarg (BM) er usammenhengende. Dette gjør også vurdere restsykdom etter terapi eller detektering av vekst resistens mutasjoner i løpet av behandlingen vanskelig ved konvensjonelle sekvensering alene 2. Ved å erstatte konvensjonelle PCR med låst nukleinsyre (LNA) -mediert villtype-blokkerende PCR (WTB-PCR) i Sanger-sekvensering, følsomheter på opp til 0,1% mutant allel i en bakgrunn av WT kan oppnås 2, 3, 4. IWTB-PCR, er berikelse for mutante alleler oppnås via tilsetning av et kort (~ 10 - 14 NT) blokkering (LNA) oligonukleotid som bindes fortrinnsvis til WT-DNA for derved å forhindre forsterkning av WT-DNA. Den mutante anrikede WTB-PCR-produktet kan deretter bli sekvensert. Ved å blokkere WT-DNA i stedet for å selektere for spesifikke mutasjoner WTB-PCR tillater anrikning av både kjente og ukjente mutasjoner er tilstede i små cellefraksjoner. Flere fremgangsmåter er for tiden benyttes for å påvise mutasjoner i små celler fraksjoner. Dette omfatter allel-spesifikk PCR-amplifikasjon-ildfast mutasjon system (ARMS), denaturerende væskekromatografi med høy ytelse (DHPLC), perler, emulsjoner, forsterkning, og Magnetics (strålte), elektrisk felt-indusert frigjøring og måling (EFIRM), høyoppløsnings smeltepunkt og andre. Imidlertid er de fleste av disse fremgangsmåter begrenset av falsk-positive og evnen til bare å påvise en mutasjon som analysen ble konstruert for fire </sup&gt;. WTB-PCR, men tillater brukeren å visualisere sekvense spor som muliggjør påvisning av flere mutasjonstyper og kan hjelpe til med å utelukke falske positive på grunn av gjenstander eller deaminering hendelser. Neste generasjon sekvensering (NGS) kan tilby et egnet alternativ til konvensjonell sekvensering, er imidlertid betydelig større kostnader, kompleksitet og lengre analysetiden gjør det unødvendig alternativ for mange sykdomstyper med noen distinkte molekylære markører eller for å overvåke pasienter under behandling for vekst resistensmutasjoner. Videre høye falske positive når påvisnings varianter med muterte allel frekvenser på mindre enn 5% kan utgjøre et problem for amplikonet NGS-baserte 5, 6. Her viser vi økningen i følsomhet oppnås ved WTB-PCR i screening for mutasjoner i den myeloide differensieringsfaktor 88-genet, som beskrevet ved Albitar et al. 3 MyD88 mutations er viktige diagnostiske og prognostiske faktorer i Waldenstrøms makroglobulinemi (WM), IgM monoklonal gammopati av ukjent betydning (IgM-MGUS), miltens marginalsone lymfom (SMZL), og diffus stor B-celle lymfom (DLBCL). MyD88 mutasjoner er funnet i nesten alle tilfeller av WM og ca 50% av pasientene med immunglobulin M (IgM) -secreting MGUS. I kontrast til dette, er MyD88 mutasjoner funnet i bare 0-6% av pasientene med SMZL og er fraværende i multippel myelom 7, 8. Fordi overlappende morfologiske, immunophenotypic, cytogenetisk, og kliniske karakteristika mellom WM og SMZL eller IgM-multippelt myelom kan ofte komplisere differensialdiagnoser, kan tilstedeværelsen av en mutasjon MyD88 tjene som en nyttig å identifisere faktor 9. MyD88 mutasjoner har også vært forbundet med større sykdomsbyrde hos pasienter med DLBCL og dårlig total overlevelse etter behandlingen 7, </sup&gt; 10. I tillegg, fordi MyD88 mutasjoner er mer hyppig finnes i aktiverte B-celle-lignende (ABC) DLBCL enn germinalsenter B-celle-lignende (GCB) DLBCL eller primær mediastinalt B-celle lymfom (PMBL), kan MyD88 mutasjons status tjene som en surrogatmarkør for ABC-subtype 11, 12. Den detaljerte protokoll som fremlegges her tjener som en mal som nye analyser kan utvikles eller de fleste eksisterende sekvenseringsanalyser kan enkelt tilpasses for nøyaktig bestemmelse av lavfrekvens-mutasjoner i forskjellige prøvetyper. Tilnærmingen kan også brukes for å overvåke og detektere motstandsdyktig mutasjoner som kan utvikle seg i tumorer eller bakterier som kan utvikle seg, mens pasienter som behandles med målrettet terapi eller antibiotika. Videre omhandler det og remedier mange av problemene forbundet med mutasjon berikelse, spesielt i formalinfiksert parafin-embedded (FFPE) vev.

<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

Presis deteksjon og identifikasjon av lavfrekvens-mutasjoner kan være problematisk ved vurdering av restsykdom etter behandling, screening for vekst resistensmutasjonene under behandling, eller når pasientene har noen sirkulerende tumorceller. Vill-type blokkerende PCR etterfulgt av sekvensanalyse gir høy følsomhet, fleksibilitet og enkelhet som en metode for å påvise disse lavfrekvente mutasjoner. Ved å legge til en spesialdesignet låst nukleinsyre oligonukleotid til en ny eller tidligere etablert konvensjonell PCR-sekvenseringsanalyse, kan følsomheten til omtrent 1 mutant allel i en bakgrunn av 1000 WT alleler bli oppnådd (1: 1000). Sekvenserings gjenstander forbundet med deaminering hendelser som vanligvis finnes i formalinfikserte paraffininnstøpte vev kan delvis avhjelpes ved bruk av uracil-DNA glycosylase under ekstraksjonstrinnene. Den optimaliserte protokollen her er spesifikk for påvisning av MyD88 mutasjon, men kan tjene som en mal for å konstruere et hvilket som helstWTB-PCR-analyse. Fordeler med den WTB-PCR-analyse i forhold til andre vanlig anvendte analyser for påvisning av lavfrekvens-mutasjoner, inkludert allele spesifikk PCR og real-time kvantitativ PCR omfatter færre tilfeller av falske positiver, større fleksibilitet og enkel implementering, og evnen til å detektere både kjente og ukjente mutasjoner.

Et begrepsmessig oversikt over WTB-PCR under utvidelsen er presentert i figur 1. Fordi et enkelt nukleotid mistilpasning i blokkerings-DNA hybrid sterkt reduserer dets smeltetemperatur (aT m = 20 - 30 ° C), forsterkning av WT allelet er sperret mens mutant templat-DNA er fri til å fullføre forlengelsen 17. På denne måte blir mutant-DNA amplifiseres eksponentielt, mens WT-DNA blir amplifisert lineært. <p class="jove_content" fo...

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.

Villtype Blokkering PCR Kombinert med direkte sekvensering som en svært følsom metode for påvisning av lavfrekvent somatiske mutasjoner

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.

Research

JoVE Journal

Genetics

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Påvisning av Kopier nummer Endringer Bruke enkelt celle Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

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Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Eneste dråpe digitale polymerasekjedereaksjons omfattende og samtidige deteksjon av mutasjoner i Hotspot regioner

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Digital PCR-basert konkurransedyktig indeks for høy-produksjon analyse av Anvendelighet inne Salmonella

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