INSTRUCTION MANUAL
EZ DNA Methylation-Lightning™ Kit Catalog Nos. D5030T, D5030 & D5031
Highlights
∙Fastest method for complete bisulfite conversion of DNA for methylation analysis.
∙Ready-to-u conversion reagent is added directly to DNA.
∙High-yield, converted DNA is ideal for PCR, MSP, array, bisulfite and Next-Gen quencing.
Contents
Product Contents (1)
Introduction to DNA Methylation (2)
Product Description (3)grateful
Product Specifications (4)
Reagent Preparation (4)
Protocol (5)
Appendix (6)
FAQs (7)
Ordering Information (8)
List of Related Products (9)
For Rearch U Only Ver. 1.0.4
Page 1
Product Contents:
Note - Integrity of kit components is guaranteed for one year from date of purcha. Reagents are routinely tested on a
lot-to-lot basis to ensure they provide maximal performance and reliability.
* The Lightning Conversion Reagent is in a ready-to-u liquid format. The reagent should be stored tightly capped at
room temperature with minimum exposure to light.
** Add 24 ml of 100% ethanol to the 6 ml M-Wash Buffer concentrate (D5030) or 96 ml of 100% ethanol to the 24 ml M-
Wash Buffer concentrate (D5031) before u. M-DNA Wash Buffer included with D5030T is supplied ready-to-u and
does not require the addition of ethanol prior to u.
EZ DNA Methylation-Lightning™ Kit technologies are patent pending.
U of Methylation Specific PCR (MSP) is protected by US Patents 5,786,146 & 6,017,704 & 6,200,756 & 6,265,171 and
International Patent WO 97/46705. No licen under the patents to u the MSP process is conveyed expressly or by
implication to the purchar by the purcha of this product.
Note - ™ Trademarks of Zymo Rearch Corporation. This product is for rearch u only and should only be ud by
trained professionals. Some reagents included with this kit are irritants. Wear protective gloves and eye protection.
Follow the safety guidelines and rules enacted by your rearch institution or facility. Freedom EVO® is a registered
trademark of Tecan Group Ltd. Pyroquencing® is a registered trademark of Biotage.
Page 2
generalmanagerIntroduction to DNA Methylation:
Cytosine methylation is a naturally occurring ba modification, in both prokaryotic
and eukaryotic organisms, consisting of the addition of a methyl group to the fifth carbon
position of the cytosine pyrimidine ring via a methyltransfera enzyme (1). In
prokaryotes DNA methylation provides a way to protect host DNA from digestion by
restriction endonucleas that are designed to eliminate foreign DNA. DNA methylation
in higher eukaryotes functions in the regulation/control of gene expression (2).
The majority of DNA methylation in mammals occurs in 5′-CpG-3′dinucleotides,
although other patterns do exist. About 80 percent of all 5′-CpG-3′dinucleotides in
piabmammalian genomes are found to be methylated, and the majority of the twenty percent
that remain unmethylated are within promoters or in the first exons of genes. It has been
demonstrated that aberrant DNA methylation is a widespread phenomenon in cancer
and may be among the earliest changes to occur during oncogenesis (3). DNA
methylation has also been shown to play a central role in gene imprinting, embryonic
development, X-chromosome gene silencing, and cell cycle regulation.
The ability to detect and quantify DNA methylation efficiently and accurately has
become esntial for the study of cancer, gene expression, genetic dias, and many
other important aspects of biology. To date, a number of methods have been developed
to detect/quantify DNA methylation including: high-performance capillary electrophoresis
(4) and methylation-nsitive arbitrarily primed PCR (5). However, the most common
techniques ud today still rely on bisulfite conversion (6).
speakoutTreating DNA with bisulfite chemically modifies non-methylated cytosines into uracil,
amapmethylated cytosines remain unchanged. Once converted, the methylation profile of the
DNA can be determined using the desired downstream application. For single locus
analysis, the region of interest is generally amplified following bisulfite conversion (i.e.,
bisulfite PCR) and then quenced or procesd for Pyroquencing®. Recent
advances in methylation detection also allow the investigation of genome-wide
methylation patterns using technologies including array-bad methods, reduced
napareprentation bisulfite quencing (RRBS), and whole genome bisulfite quencing (7).
DNA quencing results following bisulfite treatment.DNA with methylated C at
nucleotide position #5 was procesd using the EZ DNA Methylation™Kit. The
recovered DNA was amplified by PCR and then quenced directly. The methylated
cytosine at position #5 remains intact while the unmethylated cytosines at positions #7, 9,
11, 14 and 15 are completely converted into uracil following bisulfite treatment (detected
as thymine following PCR). References:
1. Adams RL. Bioessays. 1995; 17(2): 139-145.
2. Costello JF, Plass CJ. Med. Genet. 2001; 38(5): 285-30
truck是什么意思3.
3. Stirzaker C. Cancer Res. 1997; 57(11): 2229-2237.
4. Fraga MF, et al. Electrophoresis. 2000;
21(14): 2990-2994.
5. Gonzalgo ML. Cancer Res. 1997; 57(4): 594-599.
6. Frommer M. Proc. Natl. Acad. Sci. USA. 1992; 89(5): 1827-1831.
example是什么意思
7. Rakyan VK, et al. Nat. Rev. 2011, 12(8): 529-541.
Page 3 Product Description:
The EZ DNA Methylation-Lightning ™ Kit features rapid and reliable bisulfite treatment and conversion of DNA for methylation analysis. Key to the fast workflow is the ready-to-u Lightning Conversion Reagent . No preparation is necessary, simply add this unique reagent to a DNA sample, wait about an hour, and let the reaction proceed to completion. DNA denaturation and bisulfite conversion process are combined with added heat to facilitate rapid denaturation. Desulphonation and clean-up of the converted DNA is performed using a unique low-elution spin column. High yield, converted DNA is ideal for PCR, array, bisulfite and next generation quencing, etc.
Methylation Plot From Reduced Reprentation Bisulfite Sequencing (RRBS). Data shows the relative percentage of methylation at individual CpG sites in mou DNA. Methylation percentage is shown across a ~3 Mb region of mou chromosome 19. Bisulfite quencing libraries were prepared using mou genomic DNA prepped with the Genomic Clean & Concentrator ™ (D4010, D4011 – Zymo Rearch) and bisulfite converted using EZ DNA Methylation™ technology prior to Next-Gen quencing.
Zymo-Spin ™ IC Design Characteristics. The image above shows the unique design of the Zymo column that facilitates extremely small elution volumes (≥10 µl) without buffer carryover. This is unlike other columns that can retain liquid (binding/wash buffer residue) leading to carryover into the eluate.
Note: 96-Well spin -plate formats are available for processing larger numbers of samples. Also, MagPrep kits are available (p. 8) for adaptation to liquid handling robots (e.g., Tecan – Freedom EVO ®) and
automated sample prep.
Select Citations:
1. Ehrich M, et al. Nuc. Acids Res. 2007; 35 (5): e29
2. Kaneda M, et al. Nature. 2004; 429: 900-903
3. Zhang F, et al. Proc. Natl. Acad. Sci. USA. 2007; 104 (11): 4395-4400.
4. Oda M, et al. Genes & Dev. 2006; 20: 3382-3394.
5. England RPM, et al. Nature Meth. 2005; 2: 1-2.
6. Berman BP, et al. Nature Gen. 2012; 44: 40-46.
7. Leung DC, et al. Proc. Natl. Acad. Sci. USA. 2011; 108 (14): 5718-5723.
8. Heslink AT, et al. Clin. Cancer Res. 2011; 17: 2459-2465.
9. Campan M, et al. PLoS ONE. 2011, 6 (12): e28141.
Zymo-Spin™ Column Q Spin Column
Page 4
Specifications:
∙DNA Input:Samples containing between 100 pg to 2 µg of DNA. For optimal
results, the amount of input DNA should be from 200 to 500 ng.
∙Conversion Efficiency: > 99.5% of non-methylated C residues are converted to
U; > 99.5% protection of methylated cytosines.
∙DNA Recovery: >80%
Reagent Preparation:
∙Preparation of M-Wash Buffer
Add 24 ml of 100% ethanol to the 6 ml M-Wash Buffer concentrate (D5030) or 96 ml
of 100% ethanol to the 24 ml M-Wash Buffer concentrate (D5031) before u.
哥伦布传M-DNA Wash Buffer included with D5030S & D5030T is supplied ready-to-u and
2013年12月六级听力答案
does not require the addition of ethanol prior to u.
Lightning Conversion
Reagent
Overview of Bisulfite Conversion. Steps 1 and 2 occur during bisulfite
conversion, while Step 3 is performed as the DNA is bound to the column
matrix. For the reaction to proceed to completion, it is esntial the DNA be
fully denatured.
