466:. Methylated-specific primers are used, and a methylated-specific fluorescence reporter probe is also used that anneals to the amplified region. In alternative fashion, the primers or probe can be designed without methylation specificity if discrimination is needed between the CpG pairs within the involved sequences. Quantitation is made in reference to a methylated reference DNA. A modification to this protocol to increase the specificity of the PCR for successfully bisulfite-converted DNA (ConLight-MSP) uses an additional probe to bisulfite-unconverted DNA to quantify this non-specific amplification.
154:
425:
20:
525:. 5-Hydroxymethylcytosine converts to cytosine-5-methylsulfonate upon bisulfite treatment, which then reads as a C when sequenced. Therefore, bisulfite sequencing cannot discriminate between 5-methylcytosine and 5-hydroxymethylcytosine. This means that the output from bisulfite sequencing can no longer be defined as solely DNA methylation, as it is the composite of 5-methylcytosine and 5-hydroxymethylcytosine. The development of Tet-assisted oxidative bisulfite sequencing by
29:
699:. This is based on a multi-tiered strategy, whereby bisulfite sequencing is used to obtain high-resolution methylation profiles for a limited number of reference epigenomes, while less thorough analysis is performed on a wider spectrum of samples. This approach is intended to maximize the insight gained from a given amount of resources, as high-resolution genome-wide mapping remains a costly undertaking.
2214:
712:
5-hydroxymethylcytosine to 5-formylcytosine, which subsequently converts to uracil during bisulfite treatment. The only base that then reads as a C is 5‑methylcytosine, giving a map of the true methylation status in the DNA sample. Levels of 5‑hydroxymethylcytosine can also be quantified by measuring the difference between bisulfite and oxidative bisulfite sequencing.
473:(Mc-MSP). This method amplifies bisulfite-converted DNA with both methylated-specific and unmethylated-specific primers, and determines the quantitative ratio of the two products by comparing the differential peaks generated in a melting curve analysis. A high-resolution melting analysis method that uses both
702:
Gene-set analysis (for example using tools like DAVID and GoSeq) has been shown to be severely biased when applied to high-throughput methylation data (e.g. genome-wide bisulfite sequencing); it has been suggested that this can be corrected using sample label permutations or using a statistical model
450:
with possibly high methylation density, as increased numbers of CpG pairs in the primer increase the specificity of the assay. Placing the CpG pair at the 3'-end of the primer also improves the sensitivity. The initial report using MSP described sufficient sensitivity to detect methylation of 0.1%
196:
All subsequent DNA methylation analysis techniques using bisulfite-treated DNA is based on this report by
Frommer et al. (Figure 2). Although most other modalities are not true sequencing-based techniques, the term "bisulfite sequencing" is often used to describe bisulfite-conversion DNA methylation
564:
A major challenge in bisulfite sequencing is the degradation of DNA that takes place concurrently with the conversion. The conditions necessary for complete conversion, such as long incubation times, elevated temperature, and high bisulfite concentration, can lead to the degradation of about 90% of
261:
difference is present, bisulfite treatment frequently makes a number of C-to-T conversions in most regions of interest, and the resulting sensitivity approaches 100%. MS-SSCA also provides semi-quantitative analysis of the degree of DNA methylation based on the ratio of band intensities. However,
157:
Figure 3: DNA methylation analysis methods not based on methylation-specific PCR. Following bisulfite conversion, the genomic DNA is amplified with PCR that does not discriminate between methylated and non-methylated sequences. The numerous methods available are then used to make the discrimination
101:
that depend on the methylation status of individual cytosine residues, yielding single-nucleotide resolution information about the methylation status of a segment of DNA. Various analyses can be performed on the altered sequence to retrieve this information. The objective of this analysis is
711:
5-Methylcytosine and 5-hydroxymethylcytosine both read as a C in bisulfite sequencing. Oxidative bisulfite sequencing is a method to discriminate between 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution. The method employs a specific (Tet-assisted) chemical oxidation of
661:. One's epigenome varies with age, differs between tissues, is altered by environmental factors, and shows aberrations in diseases. Such rich epigenomic mapping, however, representing different ages, tissue types, and disease states, would yield valuable information on the normal function of
171:
to directly determine the nucleotides resistant to bisulfite conversion. Primers are designed to be strand-specific as well as bisulfite-specific (i.e., primers containing non-CpG cytosines such that they are not complementary to non-bisulfite-treated DNA), flanking (but not involving) the
213:
in the region. The ratio of C-to-T at individual sites can be determined quantitatively based on the amount of C and T incorporation during the sequence extension. The main limitation of this method is the cost of the technology. However, Pyrosequencing does well allow for extension to
428:
Figure 4: Methylation-specific PCR is a sensitive method to discriminately amplify and detect a methylated region of interest using methylated-specific primers on bisulfite-converted genomic DNA. Such primers will anneal only to sequences that are methylated, and thus containing
1807:
Niederhuth, Chad E.; Bewick, Adam J.; Ji, Lexiang; Alabady, Magdy S.; Kim, Kyung Do; Li, Qing; Rohr, Nicholas A.; Rambani, Aditi; Burke, John M.; Udall, Joshua A.; Egesi, Chiedozie; Schmutz, Jeremy; Grimwood, Jane; Jackson, Scott A.; Springer, Nathan M. (2016-09-27).
123:-wide level. All strategies assume that bisulfite-induced conversion of unmethylated cytosines to uracil is complete, and this serves as the basis of all subsequent techniques. Ideally, the method used would determine the methylation status separately for each
437:
This alternative method of methylation analysis also uses bisulfite-treated DNA but avoids the need to sequence the area of interest. Instead, primer pairs are designed themselves to be "methylated-specific" by including sequences complementing only unconverted
546:
of the DNA undergoing analysis is critical. It is important to ensure that reaction parameters such as temperature and salt concentration are suitable to maintain the DNA in a single-stranded conformation and allow for complete conversion. Embedding the DNA in
537:
Bisulfite sequencing relies on the conversion of every single unmethylated cytosine residue to uracil. If conversion is incomplete, the subsequent analysis will incorrectly interpret the unconverted unmethylated cytosines as methylated cytosines, resulting in
503:
of interest. One is complementary to the unaltered methylated sequence, and the other is complementary to the C-to-U-converted unmethylated sequence. The probes are also bisulfite-specific to prevent binding to DNA incompletely converted by bisulfite. The
551:
gel has been reported to improve the rate of conversion by keeping strands of DNA physically separate. Incomplete conversion rates can be estimated and adjusted-for after sequencing by including an internal control in the sequencing library, such as
118:
Bisulfite sequencing applies routine sequencing methods on bisulfite-treated genomic DNA to determine methylation status at CpG dinucleotides. Other non-sequencing strategies are also employed to interrogate the methylation at specific loci or at a
400:. Bisulfite treatment results in either introduction/removal of cleavage sites by C-to-U conversions or shift in fragment mass by G-to-A conversions in the amplified reverse strand. C-specific cleavage will cut specifically at all methylated
576:, the more limited the number of intact template molecules will likely be. This could lead to the failure of the PCR amplification, or the loss of quantitatively accurate information on methylation levels resulting from the limited
184:
strand. By incorporating high throughput sequencing adaptors into the PCR primers, PCR products can be sequenced with massively parallel sequencing. Alternatively, and labour-intensively, PCR product can be cloned and sequenced.
208:
has also been used to analyze bisulfite-treated DNA without using methylation-specific PCR. Following PCR amplification of the region of interest, pyrosequencing is used to determine the bisulfite-converted sequence of specific
672:
technology. It is believed that failures to produce cloned animals with normal viability and lifespan result from inappropriate patterns of epigenetic marks. Also, aberrant methylation patterns are well characterized in many
353:
A recently described method by Ehrich et al. further takes advantage of bisulfite-conversions by adding a base-specific cleavage step to enhance the information gained from the nucleotide changes. By first using in vitro
646:. This epigenomic information will be important in understanding how the function of the genetic sequence is implemented and regulated. Since the epigenome is less stable than the genome, it is thought to be important in
306:. DNA is bisulfite-converted, and bisulfite-specific primers are annealed to the sequence up to the base pair immediately before the CpG of interest. The primer is allowed to extend one base pair into the C (or T) using
290:, determines the rapidity of melting and consequent release of the dye. This method allows direct quantitation in a single-tube assay, but assesses methylation in the amplified region as a whole rather than at specific
23:
Figure 1: Outline of bisulfite conversion of sample sequence of genomic DNA. Nucleotides in blue are unmethylated cytosines converted to uracils by bisulfite, while red nucleotides are 5-methylcytosines resistant to
172:
methylation site of interest. Therefore, it will amplify both methylated and unmethylated sequences, in contrast to methylation-specific PCR. All sites of unmethylated cytosines are displayed as
1255:
Uhlmann K, Brinckmann A, Toliat MR, Ritter H, Nürnberg P (December 2002). "Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis".
396:
when cytosine-specific (C-specific) cleavage is desired, and incorporating dCTP when uracil-specific (U-specific) cleavage is desired. The cleaved fragments can then be analyzed by
257:. In MS-SSCA, this is used to distinguish between bisulfite-treated, PCR-amplified regions containing the CpG sites of interest. Although SSCA lacks sensitivity when only a single
580:
of template molecules. Thus, it is important to assess the amount of DNA degradation resulting from the reaction conditions employed, and consider how this will affect the desired
446:
converted from unmethylated cytosines. Methylation is determined by the ability of the specific primer to achieve amplification. This method is particularly useful to interrogate
618:
A final concern is that bisulfite treatment greatly reduces the level of complexity in the sample, which can be problematic if multiple PCR reactions are to be performed (2006).
138:
Methodologies to analyze bisulfite-treated DNA are continuously being developed. To summarize these rapidly evolving methodologies, numerous review articles have been written.
2170:
Booth MJ, Branco MR, Ficz G, Oxley D, Krueger F, Reik W, et al. quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science.
97:
residues unaffected. Therefore, DNA that has been treated with bisulfite retains only methylated cytosines. Thus, bisulfite treatment introduces specific changes in the
253:(SNP) analysis. SSCA differentiates between single-stranded DNA fragments of identical size but distinct sequence based on differential migration in non-denaturating
556:
DNA (which is known to be unmethylated) or by aligning bisulfite sequencing reads to a known unmethylated region in the organism, such as the chloroplast genome.
1525:, Molloy P (June 2002). "Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives".
721:
145:(PCR) performed under non-methylation-specific conditions (Figure 3). Microarray-based methods use PCR based on non-methylation-specific conditions also.
1597:"Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection"
488:-based methods are a logical extension of the technologies available to analyze bisulfite-treated DNA to allow for genome-wide analysis of methylation.
1300:"An analytical method for the detection of methylation differences at specific chromosomal loci using primer extension and ion pair reverse phase HPLC"
521:
Bisulfite sequencing is used widely across mammalian genomes, however complications have arisen with the discovery of a new mammalian DNA modification
565:
the incubated DNA. Given that the starting amount of DNA is often limited, such extensive degradation can be problematic. The degradation occurs as
246:
128:
342:
338:
333:
analysis to differentiate between the two polymorphic primer extension products can be used, in essence, based on the GOOD assay designed for
816:
635:
455:. In general, MSP and its related protocols are considered to be the most sensitive when interrogating the methylation status at a specific
1208:"Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE)"
141:
The methodologies can be generally divided into strategies based on methylation-specific PCR (MSP) (Figure 4), and strategies employing
1737:
Yu, M., Hon, G. C., Szulwach, K. E., Song, C., Jin, P., Ren, B., He, C. Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine.
1126:
1109:
739:
Chatterjee A, Stockwell PA, Rodger EJ and
Morison IM 2012. Comparison of alignment software for genome-wide bisulphite sequence data.
408:
within the region, rather than determining the extent of methylation of the region as a whole. This method demonstrated efficacy for
274:
A further method to differentiate converted from unconverted bisulfite-treated DNA is using high-resolution melting analysis (HRM), a
132:
1159:"Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation"
508:
is one such assay that applies the bisulfite sequencing technology on a microarray level to generate genome-wide methylation data.
1560:
Akey DT, Akey JM, Zhang K, Jin L (October 2002). "Assaying DNA methylation based on high-throughput melting curve approaches".
103:
2191:
404:. By analyzing the sizes of the resulting fragments, it is possible to determine the specific pattern of DNA methylation of
303:
250:
226:
1110:"Methylation-sensitive, single-strand conformation analysis (MS-SSCA): A rapid method to screen for and analyze methylation"
1724:
Huang Y, Pastor WA, Shen Y, Tahiliani M, Liu DR, Rao A. The
Behaviour of 5-Hydroxymethylcytosine in Bisulfite Sequencing.
167:
The first reported method of methylation analysis using bisulfite-treated DNA utilized PCR and standard dideoxynucleotide
2204:
2244:
647:
543:
928:"A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands"
1709:
Kriaucionis S, Heintz N. The nuclear DNA base 5-hydroxymethylcytosine is present in
Purkinje neurons and the brain.
634:-wide scale, where, previously, global measure of DNA methylation was feasible only using other techniques, such as
2234:
1349:"Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry"
619:
505:
489:
32:
Figure 2: Outline of the chemical reaction that underlies the bisulfite-catalyzed conversion of cytosine to uracil.
433:
that are resistant to conversion by bisulfite. In alternative fashion, unmethylated-specific primers can be used.
570:
542:
results for methylation. Only cytosines in single-stranded DNA are susceptible to attack by bisulfite, therefore
529:
at the
University of Chicago is now able to distinguish between the two modifications at single base resolution.
409:
215:
142:
2092:
424:
377:
355:
153:
19:
317:
A number of methods can be used to determine this C:T ratio. At the beginning, MS-SnuPE relied on radioactive
1725:
2186:
696:
522:
678:
477:
and melting analysis has been introduced, in particular, for sensitive detection of low-level methylation
470:
1028:"Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing"
229:
into the sequence of the sequencing primer, thus allowing for separate analysis of maternal and paternal
577:
695:
Large-scale epigenome mapping efforts are under way around the world and have been organized under the
591:
developed NEBNext
Enzymatic Methyl-seq an alternative enzymatic approach to minimize DNA damage.
584:. Techniques can also be used to minimize DNA degradation, such as cycling the incubation temperature.
2239:
1426:
1360:
939:
643:
1347:
Ehrich M, Nelson MR, Stanssens P, Zabeau M, Liloglou T, Xinarianos G, et al. (November 2005).
588:
496:
1329:
1280:
1139:
865:
681:
677:. Global hypomethylation results in decreased genomic stability, while local hypermethylation of
366:
311:
234:
611:, rendering subsequent PCR difficult. However, this situation can be avoided by monitoring the
2153:
2112:
2070:
2016:
1942:
1890:
1831:
1789:
1675:
1626:
1577:
1542:
1503:
1454:
1410:
1388:
1321:
1272:
1237:
1188:
1131:
1090:
1049:
1008:
967:
905:
857:
822:
812:
781:
654:
330:
1694:
Conversion of 5-methylcytosine to 5-hydroxymethylcytosine inmammalian DNA by MLL partner TET1
2143:
2104:
2060:
2050:
2006:
1969:
1932:
1924:
1880:
1872:
1861:"Bisulfite genomic sequencing: systematic investigation of critical experimental parameters"
1839:
1821:
1779:
1771:
1665:
1657:
1616:
1608:
1569:
1534:
1493:
1485:
1444:
1434:
1378:
1368:
1311:
1264:
1227:
1219:
1178:
1170:
1121:
1080:
1039:
998:
957:
947:
895:
849:
804:
771:
703:
to control for differences in the numberes of CpG probes / CpG sites that target each gene.
685:
474:
463:
456:
439:
430:
275:
94:
1710:
1472:
Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, et al. (April 2000).
70:
mark, and remains the most studied. In animals it predominantly involves the addition of a
1406:
926:
Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, et al. (March 1992).
608:
493:
389:
283:
282:
are analyzed directly by temperature ramping and resulting liberation of an intercalating
254:
83:
63:
1644:
Adorján P, Distler J, Lipscher E, Model F, Müller J, Pelet C, et al. (March 2002).
1430:
1364:
943:
286:
during melting. The degree of methylation, as represented by the C-to-T content in the
2218:
1937:
1912:
1844:
1809:
1621:
1596:
1522:
1383:
1348:
1183:
1158:
987:"Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites"
630:
The advances in bisulfite sequencing have led to the possibility of applying them at a
539:
363:
334:
325:
can also be used. However, matrix-assisted laser desorption ionization/time-of-flight (
322:
307:
205:
168:
55:
2065:
2038:
1784:
1759:
1670:
1645:
1538:
1498:
1473:
1232:
1207:
2228:
1885:
1860:
1449:
1414:
962:
927:
600:
2148:
2131:
1646:"Tumour class prediction and discovery by microarray-based DNA methylation analysis"
1284:
1143:
869:
28:
2132:"Gene-set analysis is severely biased when applied to genome-wide methylation data"
1760:"A modified and improved method for bisulphite based cytosine methylation analysis"
1415:"Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands"
1333:
1067:
Wong HL, Byun HM, Kwan JM, Campan M, Ingles SA, Laird PW, Yang AS (December 2006).
566:
553:
98:
71:
2108:
1961:
803:. Advances in Experimental Medicine and Biology. Vol. 544. pp. 197–204.
622:
design is more difficult, and inappropriate cross-hybridization is more frequent.
462:
The MethyLight method is based on MSP, but provides a quantitative analysis using
2130:
Geeleher P, Hartnett L, Egan LJ, Golden A, Raja Ali RA, Seoighe C (August 2013).
2055:
1973:
808:
688:. Specific patterns of methylation are indicative of specific cancer types, have
447:
59:
2213:
2171:
1913:"A new method for accurate assessment of DNA quality after bisulfite treatment"
1693:
1419:
Proceedings of the
National Academy of Sciences of the United States of America
1353:
Proceedings of the
National Academy of Sciences of the United States of America
932:
Proceedings of the
National Academy of Sciences of the United States of America
840:
Laird PW (April 2003). "The power and the promise of DNA methylation markers".
607:
residues due to inadequate alkalization of the solution. This may inhibit some
266:
as a whole in the region of interest rather than individual methylation sites.
2093:"A blueprint for a Human Epigenome Project: the AACR Human Epigenome Workshop"
1826:
662:
604:
599:
A potentially significant problem following bisulfite treatment is incomplete
485:
302:
MS-SnuPE employs the primer extension method initially designed for analyzing
258:
190:
186:
79:
67:
1835:
1775:
1223:
642:
is seen by many scientists as the logical follow-up to the completion of the
158:
based on the changes within the amplicon as a result of bisulfite conversion.
2011:
1994:
1876:
1439:
1373:
689:
639:
397:
326:
279:
181:
107:
47:
2157:
2116:
2074:
2020:
1946:
1894:
1679:
1661:
1630:
1581:
1573:
1546:
1507:
1489:
1392:
1325:
1276:
1268:
1192:
1135:
1094:
1069:"Rapid and quantitative method of allele-specific DNA methylation analysis"
1053:
1012:
952:
909:
861:
826:
785:
2197:
1928:
1793:
1458:
1241:
971:
392:, base-specificity is achieved by adding incorporating cleavage-resistant
1612:
1174:
1127:
10.1002/(SICI)1098-1004(199910)14:4<289::AID-HUMU3>3.0.CO;2-A
900:
887:
581:
573:
526:
500:
413:
405:
401:
291:
287:
263:
210:
75:
469:
Further methodology using MSP-amplified DNA analyzes the products using
321:
as the reporter of the primer extension. Fluorescence-based methods or
1316:
1299:
674:
669:
548:
443:
381:
370:
177:
173:
1692:
Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, BrudnoY, et al.
1085:
1068:
1044:
1027:
1003:
986:
776:
759:
658:
631:
569:
resulting in random strand breaks. Therefore, the longer the desired
452:
230:
124:
120:
90:
1810:"Widespread natural variation of DNA methylation within angiosperms"
853:
668:
Direct benefits of epigenomic mapping include probable advances in
298:
Methylation-sensitive single-nucleotide primer extension (MS-SnuPE)
241:
Methylation-sensitive single-strand conformation analysis (MS-SSCA)
318:
278:-based technique initially designed to distinguish SNPs. The PCR
27:
1474:"MethyLight: a high-throughput assay to measure DNA methylation"
393:
176:
in the resulting amplified sequence of the sense strand, and as
615:
of the solution to ensure that desulfonation will be complete.
345:) has also been used to distinguish primer extension products.
985:
Colella S, Shen L, Baggerly KA, Issa JP, Krahe R (July 2003).
665:
marks as well as the mechanisms leading to aging and disease.
657:, however, since the epigenome is much more variable than the
442:, or, on the converse, "unmethylated-specific", complementing
385:
374:
359:
89:
Treatment of DNA with bisulfite converts cytosine residues to
51:
1595:
Kristensen LS, Mikeska T, Krypuy M, Dobrovic A (April 2008).
692:
value, and can help to guide the best course of treatment.
612:
760:"DNA methylation: a profile of methods and applications"
314:, and the ratio of C to T is determined quantitatively.
127:. Alternative methods to bisulfite sequencing include
369:
site to the PCR primer in the initial amplification),
2202:
653:Epigenomic mapping is inherently more complex than
110:) resulting from bisulfite conversion (Figure 1).
233:. This technique is of particular usefulness for
1911:Ehrich M, Zoll S, Sur S, van den Boom D (2007).
799:El-Maarri O (2003). "Methods: DNA Methylation".
416:in multiple tissues in a cost-efficient manner.
225:, uses allele-specific primers that incorporate
16:Lab procedure detecting 5-methylcytosines in DNA
2032:
2030:
221:A variant of this technique, described by Wong
189:methods can be used to enhance the product for
1298:Matin MM, Baumer A, Hornby DP (October 2002).
881:
879:
626:Applications: genome-wide methylation analysis
1906:
1904:
1859:Grunau C, Clark SJ, Rosenthal A (July 2001).
801:Peroxisomal Disorders and Regulation of Genes
560:Degradation of DNA during bisulfite treatment
102:therefore reduced to differentiating between
8:
2086:
2084:
1995:"The necessity of a human epigenome project"
1758:Olek A, Oswald J, Walter J (December 1996).
722:Reduced representation bisulfite sequencing
149:Non-methylation-specific PCR based methods
2147:
2091:Jones PA, Martienssen R (December 2005).
2064:
2054:
2039:"Human epigenome project--up and running"
2010:
1936:
1884:
1843:
1825:
1783:
1669:
1620:
1497:
1448:
1438:
1382:
1372:
1315:
1231:
1182:
1125:
1084:
1043:
1002:
961:
951:
921:
919:
899:
775:
753:
751:
749:
412:, allowing for interrogation of numerous
423:
152:
129:Combined Bisulphite Restriction Analysis
18:
2209:
1108:Bianco T, Hussey D, Dobrovic A (1999).
886:Callinan PA, Feinberg AP (April 2006).
758:Fraga MF, Esteller M (September 2002).
732:
247:single-strand conformation polymorphism
1026:Tost J, Dunker J, Gut IG (July 2003).
339:high-performance liquid chromatography
270:High resolution melting analysis (HRM)
262:this method is designed to assess all
1720:
1718:
888:"The emerging science of epigenomics"
636:Restriction landmark genomic scanning
249:analysis (SSCA) method developed for
82:, and is implicated in repression of
7:
2192:Human Epigenome Project (HEP) - Data
388:specifically at cytosine and uracil
2198:The Epigenome Network of Excellence
1409:, Graff JR, Myöhänen S, Nelkin BD,
1206:Gonzalgo ML, Jones PA (June 1997).
133:methylated DNA immunoprecipitation
14:
894:. 15 Spec No 1 (90001): R95-101.
2212:
349:Base-specific cleavage/MALDI-TOF
197:analysis techniques in general.
2194:— by the Sanger Institute
1960:Gautreau, Isabel (2014-09-29).
1157:Wojdacz TK, Dobrovic A (2007).
358:of the region of interest into
304:single-nucleotide polymorphisms
227:single-nucleotide polymorphisms
104:single nucleotide polymorphisms
1711:Science.2009;324(5929):929-30.
707:Oxidative bisulfite sequencing
420:Methylation-specific PCR (MSP)
251:single-nucleotide polymorphism
1:
2187:Bisulfite conversion protocol
2149:10.1093/bioinformatics/btt311
2109:10.1158/0008-5472.CAN-05-3865
1962:"NEBNext End Prep Mixture v1"
1539:10.1016/S1046-2023(02)00062-2
648:gene-environment interactions
78:residues of the dinucleotide
2056:10.1371/journal.pbio.0000082
2037:Bradbury J (December 2003).
1974:10.17504/protocols.io.cg4tyv
809:10.1007/978-1-4419-9072-3_23
492:are designed using pairs of
380:at base-specific sites. As
245:This method is based on the
74:to the carbon-5 position of
58:to determine the pattern of
638:. The mapping of the human
490:Oligonucleotide microarrays
2261:
506:Illumina Methylation Assay
373:can be used to cleave the
337:. Ion pair reverse-phase
1827:10.1186/s13059-016-1059-0
684:often accounts for their
410:high-throughput screening
216:high-throughput screening
143:polymerase chain reaction
66:was the first discovered
1993:Esteller M (June 2006).
1726:PLOS ONE.2010;5(1):e8888
892:Human Molecular Genetics
481:Microarray-based methods
84:transcriptional activity
1700:. 2009;324(5929):930-5.
1440:10.1073/pnas.93.18.9821
1374:10.1073/pnas.0507816102
770:(3): 632, 634, 636–49.
697:Human Epigenome Project
523:5-hydroxymethylcytosine
517:5-Hydroxymethylcytosine
1917:Nucleic Acids Research
1865:Nucleic Acids Research
1776:10.1093/nar/24.24.5064
1764:Nucleic Acids Research
1650:Nucleic Acids Research
1601:Nucleic Acids Research
1574:10.1006/geno.2002.6851
1478:Nucleic Acids Research
1269:10.1002/elps.200290023
1224:10.1093/nar/25.12.2529
1212:Nucleic Acids Research
1163:Nucleic Acids Research
953:10.1073/pnas.89.5.1827
842:Nature Reviews. Cancer
741:Nucleic Acids Research
679:tumour suppressor gene
471:melting curve analysis
434:
159:
33:
25:
2012:10.1093/carcin/bgl033
1877:10.1093/nar/29.13.e65
533:Incomplete conversion
427:
156:
44:bisulphite sequencing
31:
22:
2172:2012;336(6083):934-7
1662:10.1093/nar/30.5.e21
1521:Rand K, Qu W, Ho T,
1490:10.1093/nar/28.8.e32
644:Human Genome Project
497:hybridization probes
2245:Genomics techniques
1929:10.1093/nar/gkl1134
1431:1996PNAS...93.9821H
1365:2005PNAS..10215785E
944:1992PNAS...89.1827F
589:New England Biolabs
1613:10.1093/nar/gkn113
1413:(September 1996).
1317:10.1002/humu.10118
1175:10.1093/nar/gkm013
901:10.1093/hmg/ddl095
435:
312:dideoxynucleotides
235:genomic imprinting
160:
34:
26:
2235:Molecular biology
1086:10.2144/000112305
1045:10.2144/03351md02
1004:10.2144/03351md01
818:978-0-306-48174-1
777:10.2144/02333rv01
655:genome sequencing
440:5-methylcytosines
431:5-methylcytosines
331:mass spectrometry
180:in the amplified
163:Direct sequencing
2252:
2217:
2216:
2208:
2175:
2168:
2162:
2161:
2151:
2127:
2121:
2120:
2088:
2079:
2078:
2068:
2058:
2034:
2025:
2024:
2014:
1990:
1984:
1983:
1981:
1980:
1957:
1951:
1950:
1940:
1908:
1899:
1898:
1888:
1856:
1850:
1849:
1847:
1829:
1804:
1798:
1797:
1787:
1755:
1749:
1735:
1729:
1722:
1713:
1707:
1701:
1690:
1684:
1683:
1673:
1641:
1635:
1634:
1624:
1592:
1586:
1585:
1557:
1551:
1550:
1518:
1512:
1511:
1501:
1469:
1463:
1462:
1452:
1442:
1403:
1397:
1396:
1386:
1376:
1359:(44): 15785–90.
1344:
1338:
1337:
1319:
1295:
1289:
1288:
1252:
1246:
1245:
1235:
1203:
1197:
1196:
1186:
1154:
1148:
1147:
1129:
1105:
1099:
1098:
1088:
1064:
1058:
1057:
1047:
1023:
1017:
1016:
1006:
982:
976:
975:
965:
955:
923:
914:
913:
903:
883:
874:
873:
837:
831:
830:
796:
790:
789:
779:
755:
744:
737:
686:loss of function
475:quantitative PCR
464:quantitative PCR
276:quantitative PCR
95:5-methylcytosine
46:) is the use of
2260:
2259:
2255:
2254:
2253:
2251:
2250:
2249:
2225:
2224:
2223:
2211:
2203:
2183:
2178:
2169:
2165:
2129:
2128:
2124:
2103:(24): 11241–6.
2097:Cancer Research
2090:
2089:
2082:
2036:
2035:
2028:
1992:
1991:
1987:
1978:
1976:
1959:
1958:
1954:
1910:
1909:
1902:
1858:
1857:
1853:
1806:
1805:
1801:
1757:
1756:
1752:
1739:Nat. Protocols
1736:
1732:
1723:
1716:
1708:
1704:
1691:
1687:
1643:
1642:
1638:
1594:
1593:
1589:
1559:
1558:
1554:
1520:
1519:
1515:
1471:
1470:
1466:
1405:
1404:
1400:
1346:
1345:
1341:
1297:
1296:
1292:
1257:Electrophoresis
1254:
1253:
1249:
1218:(12): 2529–31.
1205:
1204:
1200:
1156:
1155:
1151:
1107:
1106:
1102:
1066:
1065:
1061:
1025:
1024:
1020:
984:
983:
979:
925:
924:
917:
885:
884:
877:
854:10.1038/nrc1045
839:
838:
834:
819:
798:
797:
793:
757:
756:
747:
738:
734:
730:
718:
709:
628:
609:DNA polymerases
597:
562:
535:
519:
514:
494:oligonucleotide
483:
422:
390:ribonucleotides
351:
300:
284:fluorescent dye
272:
255:electrophoresis
243:
203:
165:
151:
116:
106:(cytosines and
64:DNA methylation
54:before routine
42:(also known as
17:
12:
11:
5:
2258:
2256:
2248:
2247:
2242:
2237:
2227:
2226:
2222:
2221:
2201:
2200:
2195:
2189:
2182:
2181:External links
2179:
2177:
2176:
2163:
2142:(15): 1851–7.
2136:Bioinformatics
2122:
2080:
2026:
1999:Carcinogenesis
1985:
1952:
1900:
1851:
1814:Genome Biology
1799:
1770:(24): 5064–6.
1750:
1730:
1714:
1702:
1685:
1636:
1587:
1552:
1513:
1464:
1425:(18): 9821–6.
1398:
1339:
1304:Human Mutation
1290:
1263:(24): 4072–9.
1247:
1198:
1149:
1114:Human Mutation
1100:
1059:
1018:
977:
938:(5): 1827–31.
915:
875:
832:
817:
791:
745:
731:
729:
726:
725:
724:
717:
714:
708:
705:
627:
624:
596:
595:Other concerns
593:
561:
558:
540:false positive
534:
531:
518:
515:
513:
510:
482:
479:
421:
418:
364:RNA polymerase
362:(by adding an
350:
347:
335:SNP genotyping
323:Pyrosequencing
308:DNA polymerase
299:
296:
271:
268:
242:
239:
206:Pyrosequencing
202:
201:Pyrosequencing
199:
169:DNA sequencing
164:
161:
150:
147:
115:
112:
15:
13:
10:
9:
6:
4:
3:
2:
2257:
2246:
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2238:
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2215:
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2206:
2199:
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2188:
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2173:
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2159:
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2126:
2123:
2118:
2114:
2110:
2106:
2102:
2098:
2094:
2087:
2085:
2081:
2076:
2072:
2067:
2062:
2057:
2052:
2048:
2044:
2040:
2033:
2031:
2027:
2022:
2018:
2013:
2008:
2005:(6): 1121–5.
2004:
2000:
1996:
1989:
1986:
1975:
1971:
1967:
1963:
1956:
1953:
1948:
1944:
1939:
1934:
1930:
1926:
1922:
1918:
1914:
1907:
1905:
1901:
1896:
1892:
1887:
1882:
1878:
1874:
1871:(13): E65-5.
1870:
1866:
1862:
1855:
1852:
1846:
1841:
1837:
1833:
1828:
1823:
1819:
1815:
1811:
1803:
1800:
1795:
1791:
1786:
1781:
1777:
1773:
1769:
1765:
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1734:
1731:
1727:
1721:
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1715:
1712:
1706:
1703:
1699:
1695:
1689:
1686:
1681:
1677:
1672:
1667:
1663:
1659:
1656:(5): 21e–21.
1655:
1651:
1647:
1640:
1637:
1632:
1628:
1623:
1618:
1614:
1610:
1606:
1602:
1598:
1591:
1588:
1583:
1579:
1575:
1571:
1568:(4): 376–84.
1567:
1563:
1556:
1553:
1548:
1544:
1540:
1536:
1533:(2): 114–20.
1532:
1528:
1524:
1517:
1514:
1509:
1505:
1500:
1495:
1491:
1487:
1483:
1479:
1475:
1468:
1465:
1460:
1456:
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1446:
1441:
1436:
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1428:
1424:
1420:
1416:
1412:
1408:
1402:
1399:
1394:
1390:
1385:
1380:
1375:
1370:
1366:
1362:
1358:
1354:
1350:
1343:
1340:
1335:
1331:
1327:
1323:
1318:
1313:
1310:(4): 305–11.
1309:
1305:
1301:
1294:
1291:
1286:
1282:
1278:
1274:
1270:
1266:
1262:
1258:
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1248:
1243:
1239:
1234:
1229:
1225:
1221:
1217:
1213:
1209:
1202:
1199:
1194:
1190:
1185:
1180:
1176:
1172:
1168:
1164:
1160:
1153:
1150:
1145:
1141:
1137:
1133:
1128:
1123:
1120:(4): 289–93.
1119:
1115:
1111:
1104:
1101:
1096:
1092:
1087:
1082:
1078:
1074:
1073:BioTechniques
1070:
1063:
1060:
1055:
1051:
1046:
1041:
1037:
1033:
1032:BioTechniques
1029:
1022:
1019:
1014:
1010:
1005:
1000:
997:(1): 146–50.
996:
992:
991:BioTechniques
988:
981:
978:
973:
969:
964:
959:
954:
949:
945:
941:
937:
933:
929:
922:
920:
916:
911:
907:
902:
897:
893:
889:
882:
880:
876:
871:
867:
863:
859:
855:
851:
848:(4): 253–66.
847:
843:
836:
833:
828:
824:
820:
814:
810:
806:
802:
795:
792:
787:
783:
778:
773:
769:
765:
764:BioTechniques
761:
754:
752:
750:
746:
742:
736:
733:
727:
723:
720:
719:
715:
713:
706:
704:
700:
698:
693:
691:
687:
683:
680:
676:
671:
666:
664:
660:
656:
651:
649:
645:
641:
637:
633:
625:
623:
621:
616:
614:
610:
606:
602:
601:desulfonation
594:
592:
590:
585:
583:
579:
575:
572:
568:
567:depurinations
559:
557:
555:
550:
545:
541:
532:
530:
528:
524:
516:
511:
509:
507:
502:
498:
495:
491:
487:
480:
478:
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472:
467:
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460:
458:
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449:
445:
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411:
407:
403:
399:
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391:
387:
383:
379:
376:
372:
368:
365:
361:
357:
356:transcription
348:
346:
344:
340:
336:
332:
328:
324:
320:
315:
313:
309:
305:
297:
295:
293:
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269:
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240:
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236:
232:
228:
224:
219:
217:
212:
207:
200:
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194:
192:
188:
183:
179:
175:
170:
162:
155:
148:
146:
144:
139:
136:
134:
130:
126:
122:
113:
111:
109:
105:
100:
96:
93:, but leaves
92:
87:
85:
81:
77:
73:
69:
65:
61:
57:
53:
50:treatment of
49:
45:
41:
38:
30:
21:
2166:
2139:
2135:
2125:
2100:
2096:
2046:
2043:PLOS Biology
2042:
2002:
1998:
1988:
1977:. Retrieved
1966:protocols.io
1965:
1955:
1920:
1916:
1868:
1864:
1854:
1817:
1813:
1802:
1767:
1763:
1753:
1745:
1741:
1738:
1733:
1705:
1697:
1688:
1653:
1649:
1639:
1604:
1600:
1590:
1565:
1561:
1555:
1530:
1526:
1516:
1484:(8): 32e–0.
1481:
1477:
1467:
1422:
1418:
1401:
1356:
1352:
1342:
1307:
1303:
1293:
1260:
1256:
1250:
1215:
1211:
1201:
1166:
1162:
1152:
1117:
1113:
1103:
1079:(6): 734–9.
1076:
1072:
1062:
1038:(1): 152–6.
1035:
1031:
1021:
994:
990:
980:
935:
931:
891:
845:
841:
835:
800:
794:
767:
763:
743:40(10): e79.
740:
735:
710:
701:
694:
667:
652:
629:
617:
598:
586:
563:
554:lambda phage
544:denaturation
536:
520:
484:
468:
461:
436:
352:
316:
310:terminating
301:
273:
244:
222:
220:
204:
195:
166:
140:
137:
117:
99:DNA sequence
88:
72:methyl group
43:
39:
36:
35:
2240:Epigenetics
512:Limitations
448:CpG islands
60:methylation
40:sequencing
24:conversion.
2229:Categories
2049:(3): E82.
1979:2021-05-19
1923:(5): e29.
1820:(1): 194.
1607:(7): e42.
1169:(6): e41.
728:References
690:prognostic
663:epigenetic
605:pyrimidine
499:targeting
486:Microarray
378:transcript
259:nucleotide
237:analysis.
191:sequencing
187:Nested PCR
68:epigenetic
56:sequencing
1836:1474-760X
1411:Baylin SB
1407:Herman JG
682:promoters
640:epigenome
587:In 2020,
501:CpG sites
414:CpG sites
406:CpG sites
402:CpG sites
398:MALDI-TOF
327:MALDI-TOF
292:CpG sites
280:amplicons
264:CpG sites
218:methods.
211:CpG sites
182:antisense
135:(MeDIP).
108:thymidine
48:bisulfite
37:Bisulfite
2158:23732277
2117:16357125
2075:14691553
2021:16699174
1947:17259213
1895:11433041
1680:11861926
1631:18344521
1582:12376091
1562:Genomics
1547:12095268
1523:Clark SJ
1508:10734209
1393:16243968
1326:12325026
1285:43737807
1277:12481262
1193:17289753
1144:22814772
1136:10502775
1095:17191619
1054:12866415
1013:12866414
910:16651376
870:19574628
862:12671664
827:14713229
786:12238773
716:See also
582:amplicon
578:sampling
574:amplicon
527:Chuan He
444:thymines
384:cleaves
367:promoter
288:amplicon
178:adenines
174:thymines
76:cytosine
2219:Biology
1938:1865059
1845:5037628
1794:9016686
1748:, 2159.
1698:Science
1622:2367707
1527:Methods
1459:8790415
1427:Bibcode
1384:1276092
1361:Bibcode
1334:3178841
1242:9171109
1184:1874596
972:1542678
940:Bibcode
675:cancers
670:cloning
549:agarose
453:alleles
382:RNase A
371:RNase A
341:(IP-RP-
231:alleles
114:Methods
2205:Portal
2156:
2115:
2073:
2066:300691
2063:
2019:
1945:
1935:
1893:
1883:
1842:
1834:
1792:
1785:146326
1782:
1678:
1671:101257
1668:
1629:
1619:
1580:
1545:
1506:
1499:102836
1496:
1457:
1447:
1391:
1381:
1332:
1324:
1283:
1275:
1240:
1233:146734
1230:
1191:
1181:
1142:
1134:
1093:
1052:
1011:
970:
960:
908:
868:
860:
825:
815:
784:
659:genome
632:genome
620:Primer
319:ddNTPs
223:et al.
125:allele
121:genome
91:uracil
1886:55789
1450:38513
1330:S2CID
1281:S2CID
1140:S2CID
963:48546
866:S2CID
457:locus
2154:PMID
2113:PMID
2071:PMID
2017:PMID
1943:PMID
1891:PMID
1832:ISSN
1790:PMID
1742:2012
1676:PMID
1627:PMID
1578:PMID
1543:PMID
1504:PMID
1455:PMID
1389:PMID
1322:PMID
1273:PMID
1238:PMID
1189:PMID
1132:PMID
1091:PMID
1050:PMID
1009:PMID
968:PMID
906:PMID
858:PMID
823:PMID
813:ISBN
782:PMID
394:dTTP
343:HPLC
131:and
2144:doi
2105:doi
2061:PMC
2051:doi
2007:doi
1970:doi
1933:PMC
1925:doi
1881:PMC
1873:doi
1840:PMC
1822:doi
1780:PMC
1772:doi
1666:PMC
1658:doi
1617:PMC
1609:doi
1570:doi
1535:doi
1494:PMC
1486:doi
1445:PMC
1435:doi
1379:PMC
1369:doi
1357:102
1312:doi
1265:doi
1228:PMC
1220:doi
1179:PMC
1171:doi
1122:doi
1081:doi
1040:doi
999:doi
958:PMC
948:doi
896:doi
850:doi
805:doi
772:doi
603:of
571:PCR
451:of
386:RNA
375:RNA
360:RNA
80:CpG
52:DNA
2231::
2152:.
2140:29
2138:.
2134:.
2111:.
2101:65
2099:.
2095:.
2083:^
2069:.
2059:.
2045:.
2041:.
2029:^
2015:.
2003:27
2001:.
1997:.
1968:.
1964:.
1941:.
1931:.
1921:35
1919:.
1915:.
1903:^
1889:.
1879:.
1869:29
1867:.
1863:.
1838:.
1830:.
1818:17
1816:.
1812:.
1788:.
1778:.
1768:24
1766:.
1762:.
1744:,
1717:^
1696:.
1674:.
1664:.
1654:30
1652:.
1648:.
1625:.
1615:.
1605:36
1603:.
1599:.
1576:.
1566:80
1564:.
1541:.
1531:27
1529:.
1502:.
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