Высокая чувствительность 5-hydroxymethylcytosine Обнаружение в Balb / C ткани мозга
Summary
EpiMark 5-HMC и 5-тС Анализ Kit может быть использован для анализа и количественного 5-метилцитозин и 5-hydroxymethylcytosine в специально cific локуса. Комплект отличается 5-тС из 5-HMC добавлением глюкозы в гидроксильные группы 5-HMC через ферментативной реакции использованием β-glucosyltransferase (Т4-BGT). Когда 5-HMC происходит в контексте КСДУ, эта модификация превращает расщепляемого сайт MspI к не-расщепляемого сайта.
Abstract
DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells.
Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci.
The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol.
Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected.
This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC.
The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site.
In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.
Protocol
Discussion
Есть несколько важных вещей, чтобы рассмотреть при создании этого эксперимента. Во-первых, важно, чтобы glucosylation геномной ДНК переходит к завершению. Последовательность Специфичность Т4-BGT не известно, и это, кажется, нет другого выбора, для подложки последовательности. Поэтому в некоторых случаях более длительное время инкубации может быть необходимым. Во-вторых, MspI и HpaII пищеварения должна быть полной, чтобы избежать фонового сигнала. Для этих ферментов рестрикции, мы рекомендуем инкубации в течение 4 часов, но больше инкубации могут быть выполнены при неполном расщеплении наблюдается. В-третьих, количество входных ДНК может быть скорректирован в зависимости от доступности, с 20 NGS ДНК может быть использована для конечной точки ПЦР. Наконец, мы рекомендуем использовать поставляемые контроль ДНК, которые могут работать параллельно с геномной ДНК в течение 5-MC и 5-HMC количественной оценке.
Disclosures
The authors have nothing to disclose.
Acknowledgements
Sriharsa Прадхан, Шеннон Мори Кинни, Hang Gyeong Чин, Юрате Bitinaite, Ю. Чжэн, Пьер Оливье Esteve, Ромуальдас Vaisvila, лабораторные Стивен Э. Якобсен с, Лос-Анджелесе. Эта работа была частично поддержана NIH 1R44GM095209-01.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| EpiMark™ 5-hmC and 5-mC Analysis Kit | New England Biolabs | E3317 | ||
| Locus specific primers, flanking a CCGG site of interest | Custom per experiment; provided by user | |||
| LongAmp® Taq DNA Polymerase | New England Biolabs | M0323 | ||
| dNTPs | New England Biolabs | N0447 | ||
| molecular biology grade water |
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