词条 | Combined bisulfite restriction analysis |
释义 |
ProcedureBisulfite TreatmentGenomic DNA of interest is treated with sodium bisulfite, which introduces methylation-dependent sequence differences. During sodium bisulfite treatment, unmethylated cytosine residues are converted to uracil, while methylated cytosine residues are unaffected. PCR AmplificationBisulfite treated DNA is then PCR amplified, resulting in cytosine residues at originally methylated positions, and thymine residues at originally unmethylated position (that were converted to uracil). Primers used during this step do not contain CpG sites (the common target of cytosine methylation), so the amplificiation process does not discriminate between templates based on methylation status. PCR products are purified to ensure complete digestion in the following step. Restriction DigestThe above steps lead to the methylation dependent retention or loss of CpG-containing restriction enzyme sites, such as those for TaqI (TCGA) and BstUI (CGCG), depending on whether the cytosine residue was originally methylated or not, respectively. Due to the methylation-independent amplification in the above step, the resulting PCR products will be a mixed population of fragments that have lost or retained CpG-containing restriction enzyme sites, whose respective percentages will be directly correlated to the original level of DNA methylation in the sample DNA. PCR products are then treated with a restriction enzyme (e.g. BstUI), which will only cleave sites that were originally methylated (CGCG), while leaving sites that were originally unmethylated (TGTG). To ensure that all CpG sites are retained due to originally being methylated, and not a remnant of incomplete bisulfite conversion, a control digestion is performed, with enzymes such as Hsp92II which recognizes the sequence CATG, none of which should be remaining after bisulfite conversion (with the rare exception of non-CpG methylation) and thus no cleavage should occur if bisulfite conversion was complete. QuantificationThe digested fragments are then separated by polyacrylamide gel electrophoresis with the expected appearance of bands corresponding to a single large undigested fragment, and multiple smaller bands corresponding to digested fragments. Quantitative amount of DNA in these bands can be determined with a device such as a phosphoimager, after which the methylation percentage of the original sample can be calculated by: Usage and ApplicationsCOBRA has been used extensively in many research-based applications such as screening for DNA methylation changes at gene promoters in cancer studies,[3][4] detecting altered methylation patterns at imprinted genes,[5] and characterizing methylation patterns in the genome during development in mammals.[6][7] In medicine, COBRA has been used as a tool to help diagnose human disease involving aberrant DNA methylation. Researchers utilized COBRA in conjunction with denaturing high performance liquid chromatography in the diagnosis of the genetic imprinting disorder Russell-Silver syndrome where hypomethylation of the imprinted gene H19 is responsible for the disorder in up to 50% of patients.[8] Strengths
Weaknesses
AlternativesIn general, COBRA is often combined with other DNA methylation analyses and is frequently used in the initial screening of a loci of interest. If COBRA suggests altered methylation patterns, then more rigorous, labor-intensive techniques can be applied, such as bisulfite sequencing or MeDIP. References1. ^{{cite journal|last1= Xiong|first1= Zhenggang|last2= Laird|first2= Peter W.|year= 1997|title= COBRA: a sensitive and quantitative DNA methylation assay|journal= Nucleic Acids Research|volume= 25|issue= 12|pages= 2532–2534|doi= 10.1093/nar/25.12.2532}} {{DEFAULTSORT:Combined Bisulfite Restriction Analysis}}2. ^{{cite journal|last= Laird|first= Peter W.|year= 2003|title= The power and the promise of DNA methylation markers|journal= Nature Reviews Cancer|volume= 3|issue= April|pages= 253–266|doi= 10.1038/nrc1045|pmid=12671664}} This paper had 576 references as of February 2–11, according to Scopus 3. ^{{cite journal|last1= Shen|first1= Lanlan|year= 2005|title= MGMT Promoter Methylation and Field Defect in Sporadic Colorectal Cancer|journal= Journal of the National Cancer Institute|volume= 97|issue= 18|pages= 1330–1338|doi=10.1093/jnci/dji275|pmid=16174854|display-authors=etal|citeseerx=10.1.1.536.6096}} 4. ^{{cite journal|last1= Suter|first1= Catherine, M.|year= 2004|title= Germline epimutation in MLH1 in individuals with multiple cancers|journal= Nature Genetics|volume= 36|pages= 497–501|doi= 10.1038/ng1342|issue=5|display-authors=etal}} 5. ^{{cite journal|last1= Bliek|first1= Jet|year= 2009|title= Hypomethylation at multiple maternally methylated imprinted regions including PLAGL1 and GNAS loci in Beckwith–Wiedemann_syndrome, and characterizing DNA methylation patterns in the genome during development in mammals|journal= European Journal of Human Genetics|volume= 17|pages= 611–618|doi= 10.1038/ejhg.2008.233|issue=5|display-authors=etal|url= https://air.unimi.it/bitstream/2434/54678/2/ejhg2008233a.pdf}} 6. ^{{cite journal|last1= Wernig|first1= Marius|year= 2007|title= In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state|journal= Nature|volume= 448|pages= 318–324 | doi = 10.1038/nature05944|pmid=17554336|issue=7151|display-authors=etal}} 7. ^{{cite journal|last1= Mikkelsen|first1= Tarjei S.|year= 2008|title= Dissecting direct reprogramming through integrative genomic analysis|journal= Nature|volume= 454|issue= July|pages= 49–56|doi= 10.1038/nature07056|display-authors=etal|pmid=18509334|pmc=2754827}} 8. ^{{cite journal|last1= Hattori|first1= M.|year= 2009|title= Diagnosis of Russell-Silver syndrome by the combined bisulfite restriction analysis-denaturing high-performance liquid chromatography assay|journal= Genetic testing and molecular biomarkers|volume= 13|issue= 5|pages= 623–630|doi=10.1089/gtmb.2009.0018|display-authors=etal}} 9. ^{{cite journal|last1= Fraga|first1= Mario F.|last2= Esteller|first2= Manel|year= 2002|title= DNA Methylation: A Profile of Methods and Applications|journal= BioTechniques|volume= 33|pages= 633–649}} 1 : Molecular biology techniques |
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