Accurate DNA Methylation Analysis with Successful Bisulfite Conversion Webinar

Sample to Insight

Accurate DNA methylation analysis by successful bisulfite conversionDr. Inga Irle, Global Product Manager

Sample to Insight

Accurate DNA methylation analysis by successful bisulfite conversion

Legal Disclaimer

2

QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.

http://www.qiagen.com/

Sample to Insight

Accurate DNA methylation analysis by successful bisulfite conversion 3

Agenda

Introduction to DNA methylation1 19

Analysis of DNA methylation2 20

Challenges of bisulfite conversion3 21

Overview of QIAGEN solutions for bisulfite conversion

4 22

Summary5 23

Sample to Insight


Agenda





4 22

Summary5 23

Sample to Insight

Accurate DNA methylation analysis by successful bisulfite conversion

1) Introduction to DNA Methylation

DNA Methylation

• Addition of a methyl (CH3) group at the 5-carbon of the cytosine ring

• In humans, 5-mC is found in approximately 1.5% of gDNA

• 5-mC occurs almost exclusively in the context of paired symmetrical methylation of a CpG site

.CpG islands• In the bulk of genomic DNA, most CpG sites are heavily methylated • CpG islands remain unmethylated, thus allowing gene expression to occur • When a CpG island in the promoter region of a gene is methylated, expression of

the gene is repressed

Biological relevance• DNA methylation acts in concert with other epigenetic mechanisms to regulate

normal gene expression and facilitate chromatin organization within cells. • Abnormal methylation has been associated with a variety of diseases, e.g. cancer• The analysis of of DNA methylation could provide value insights on disease

development, diagnosis and therapy

Sample to Insight


Agenda





4 22

Summary5 23

Sample to Insight


2) Analysis of DNA methylation - Principle of bisulfite conversion

Bisulfite conversion – the key step in DNA methylation analysis

• Crucial step in methylation analysis workflow• Efficient base conversion required for accurate assessment of methylation status

Sample to Insight


2) Analysis of DNA methylation - workflow

EpiTect Fast Bisulfite Kits are compatible with various analysis methods

DNA purification

Bisulfite treatment Amplification Analysis

• QIAamp

• EpiTect Fast FFPE Bisulfite • EpiTect Fast LyseAll Bisulfite

• QIAcube • QIAsymphony

• EpiTect Fast DNA Bisulfite

• QIAcube

• EpiTect Whole Bisulfitome

• EpiTect MSP• EpiTect MethyLight PCR• EpiTect HRM• EpiTect Control DNAs• PyroMark PCR • PyroMark CpG Assays

• Rotor-Gene Q• Pyromark Q48

Kits

Inst

rum

ents

Sample to Insight


Agenda





4 22

Summary5 23

Sample to Insight


Challenges of bisulfite conversion

a) Sample preparation

b) DNA protection during bisulfite conversion

c) Complete conversion reaction

d) Fast results

e) High recovery and concentration of bsDNA for downstream analysis

DNA purification


Sample to Insight


3) Challenges of bisulfite conversion


• Samples are precious and not unlimited• Purity of the gDNA is very important for the success of complete bisulfite

conversion, as protein contamination prevent efficient conversion• DNA purification is often required before bisulfite conversion, resulting in loss of

DNA• Formalin-fixed paraffin embedded (FFPE) tissues are particularly challenging –

because of the fixing process, it causes modifications to the DNA, including DNA-protein crosslinks

Sample to Insight




• EpiTect Fast Bisulfite Conversion Kits provide a very fast, streamlined procedure for efficient bisulfite conversion and purification of DNA directly from any starting material — blood, cells, tissue samples or FFPE slices

• The kits function as an integrated system of solutions for direct bisulfite conversion of DNA from blood, cells, tissue samples or FFPE slices

• Integrated lysis minimizes loss of sample material

Sample to Insight


3) Challenges during bisulfite conversion

b) DNA protection

• Conversion requires ssDNA • Chemical conversion occurs at non-physiological pH and temperature which

causes DNA fragmentation• The more break-points, the smaller the DNA fragments• The smaller the DNA, the higher the risk that downstream applications fail• Lower analysis sensitivity would need to be compensated by increasing amount

of target DNA

Bisulfite Conversionw/o DNA protection

Detectable DNA:1/15 DNA molecules

Detectable DNA:10/15 DNA molecules

Sample to Insight


3) Challenges during bisulfite conversion

b) DNA protection

Unique DNA Protect Buffer:• Fewer break-points• Enhanced performance of downstream

applications• High sensitivity - less DNA is needed• Minimal DNA degradation ensurs high yields

of DNA that can be analyzed

Sample to Insight



c) Complete conversion

• Incomplete conversion of unmethylated cytosines leads to false positive methylation results

• Complete denaturation of the DNA is necessary for efficient bisulfite conversion• Optimal pH needed for complete conversion of cytosine to uracil

Complete conversion requires single-stranded DNA, optimum pH and temperature, as well as a well-balanced ratio of DNA to bisulfite.

• DNA Protect buffer facilitates highly effective DNA denaturation and results in the formation of single-stranded DNA that is necessary for complete cytosine conversion

• pH indicator confirms correct pH and correct mixing

Sample to Insight



c) Complete conversion

EpiTect Fast Bisulfite Conversion Kits contain an innovative Bisulfite Solution that enables ultrafast, highly efficient bisulfite conversion, even on difficult-to-analyze samples such as X-chromosome genomic DNA derived from a female sample, where one of the 2 X-chromosomes is highly methylated:

16

Sample to Insight



d) Fast results

• Competitor’s bisulfite kits contain aliquoted bisulfite salt that first needs to be dissolved and has a limited shelf life

• Furthermore, some kits require an overnight conversion process

• The EpiTect Fast Kits contain a ready-to-use bisulfite solution that can be stored at room temperature (15–25°C) for at least 6 months.

• Bisulfite conversion in as short as 30 minutes of processing time with a conversion rate <99.8%

Sample to Insight




Bisulfite treated DNA can be used for various downstream applications, e.g.:• PCR: end-point, real-time, and methylation-specific • Bisulfite sequencing (direct and cloning) • COBRA (Combined Bisulfite Restriction Analysis) • Pyrosequencing• Next Generation Sequencing

Reliable downstream analysis required sufficient amount of intact bsDNA. EpiTect Fast Kits enable accurate downstream analysis by:• ensuring optimal DNA protection during bisulfite conversion• providing highly concentrated bsDNA in as little as 10 µl Elution Buffer

Sample to Insight




• Detection technologies get more and more sensitive, but not necessarily more robust

• Time and costs wasted for the processing of low-quality samples• The quantity of bisulfite-converted DNA can be determined using a

spectrophotometer• Use a value of 40 µg/mL for A260 = 1.0, because the converted DNA more

resembles RNA• Check DNA quality by determining the A260/A280 ratio of the bisulfite converted

DNA, the ratio should be between 1.7 and 1.9

DNA purification


QC

Sample to Insight


Agenda





4 22

Summary5 23

Sample to Insight


4) Overview of QIAGEN solutions for reliable bisulfite conversion

Product Key Advantages New features

EpiTect Fast DNA Bisulfite Kit

Highly concentrated DNA suitable for all downstream applications; enhanced sensitivity of downstream applications due to minimal DNA fragmentation.

Low elution volume, ready-to-use bisulfite solution and fast conversion time combined with proven DNA protection.

EpiTect Fast LyseAll Bisulfite Kit

Minimized loss of sample material due to integrated sample lysis; high DNA concentration enables use in all downstream applications.

Integrated sample lysis (cells, tissue, blood) ready-to-use bisulfite solution and fast conversion time and low elution volume combined with proven DNA protection.

EpiTect Fast FFPE Bisulfite Kit

Highest yield, maximized conversion and highest concentration of converted DNA from as little as one FFPE slice.

Optimized DNA isolation, ready-to-use bisulfite solution and fast conversion time and low elution volume combined with proven DNA protection.

Sample to Insight


Agenda





4 22

Summary5 23

Sample to Insight


5) Summary

EpiTect Fast Bisulfite Kits

Features• Bisulfite conversion in as little as 30 minutes• Ready-to-use bisulfite conversion reagent• Unique DNA Protect Buffer to prevent DNA degradation during treatment• Integrated DNA isolation and conversion• Minimal elution volume to achieve highly concentrated bsDNA

Benefits• Methylation analysis ready in just one day, no over-night incubations – saves

time and allows faster results• No aliquoted bisulfite salt with limited shelf life, no dissolving steps – saves

preparation time, material and money• Minimal DNA degradation – ensures higher yields of DNA can be analyzed and

increases the likelihood of finding an intact DNA fragment for PCR analysis

Sample to Insight


EpiTect Fast System

Odering information

• EpiTect Fast Bisulfite Kit (10) 59802 o Universal trial kit - DNA isolation and bisulfite conversion for all kinds of samples

• EpiTect Fast DNA Bisulfite Kito EpiTect Fast DNA Bisulfite Kit (50) 59824o EpiTect Fast DNA Bisulfite Kit (200) 59826 o EpiTect Fast 96 Bisulfite Kit (2) 59720

• EpiTect Fast FFPE Bisulfite Kito EpiTect Fast FFPE Bisulfite Kit (50) 59844 o EpiTect Fast FFPE Bisulfite Kit (200) 59846o EpiTect Fast 96 FFPE Bisulfite Kit (2) 59740

• EpiTect Fast LyseAll Bisulfite Kito EpiTect Fast LyseAll Bisulfite Kit (50) 59864o EpiTect Fast LyseAll Bisulfite Kit (200) 59866 o EpiTect Fast 96 LyseAll Bisulfite Kit (2) 59760

EpiTect Fast Bisulfite System

Sample to Insight


Questions?

Thank you very much for your attention!

Dr. Inga [email protected]

Contact QIAGEN Technical ServiceCall: 1-800-426-8157 for US

Call: +49 2103-29-12400 for EU

Email: [email protected]

[email protected]

[email protected]

mailto:[email protected]

mailto:[email protected]

Accurate DNA Methylation Analysis with Successful Bisulfite Conversion Webinar

Healthcare

Transcript of Accurate DNA Methylation Analysis with Successful Bisulfite Conversion Webinar