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Epigenetics Lab
December 1, 2008
Goals of today’s lab:
1. Understand the basic molecular techniques used
in the lab to study epigenetic silencing in cancer
2. Analyze primary data and draw conclusions
3. Design DNA primers for future experiments
Restriction Sensitive Southern
Draw the bands that you would expect of
the following digests.
Lane 1: Normal tissue undigested
Lane 2: Normal tissue digested with RE1
Lane 3: Normal tissue digested with
MSRE1
Lane 4: Tumor tissue undigested
Lane 5: Tumor tissue digested with RE1
Lane 6: Tumor tissue digested with
MSRE1
1
1000
900
800
700
600
500
400
1000
0
CpG Island
750 bp
RE1 and MSRE1
cut site
300
200
100
2
3
4
5
6
Follow up questions to restriction enzyme southern
1.
What would you expect the digest of a tumor sample that
is 50% methylated to look like?
2.
Why is the non-methylation sensitive restriction digest a
necessary control?
3.
Why is a parent band digest needed?
Sodium Bisulfite Conversion
Write the following sequence after bisulfite conversion:
5’ CATCCGTTACCGTTGGGCTCGAAA 3’
1. If unmethylated
2.
If methylated
Follow-up question:
1. If you wanted to perform PCR in a methylated sensitive manner - would
you do PCR before or after sodium bisulfite conversion? Why?
Primer Design
• Take the following sequence and design a forward
(or sense) and reverse (or anti sense) primer to
amplify this sequence. Start with the base pair
that is underlined and work in the 5’ to 3’
direction. Primers must be between 7- 10 base
pairs. Underlining is fine. Make sure you annealing
temperatures match.
5’ ACAGATCTGGGTACAACTGACACGGACTTGAC 3’
3’ TGTCTAGACCCATGTTGACTGTGCCTGAACTG 5’
Remember 4 C/G + 2 A/T
Example of MSP Data
•
Why does the second sample have bands in both the
Methylated and Unmethylated lanes?
1
2
Q
TIFF (Unco
are need
Assignment Part I: Analyzing MSP Data
•
1.
After looking at the MSP data answer the following questions:
Is this gene methylated in normal tissue (if so, how often)?
2.
Is this gene methylated in tumor samples (if so, how often)?
3.
Based on the MSP data from Normal/Tumor pairs - would you
hypothesize Gene X is a TSG or an oncogene? Why?
4.
Why is normal tissue DNA methylation a good control?
MSP for Gene X
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
• Here is the data from a recent survey of 12 breast cancer
patients - looking for DNA methylation of Gene X
– N = Normal T= Tumor
– U = Unmethylated M= Methylated
Create MSP primers
• You have the DNA sequence to the
CpG island of a known TSG
– To determine the methylation status of
this gene in tumor samples you are
planning on using MSP (since you are
now an expert)
• You must design MSP primers (both
Sense and Anti-sense and
Methylated and Unmethylated)
For Homework: Criteria for MSP primers
• Each primer:
– Must encompass 3 CpGs
– Each primer must be between 20-30 bp
– Methylated S and AS primers must have the same
melting temperatures (4 G/C + 2 A/T)
– Unmethylated pair must match as well
– Account for each unmethylated Cytosines to be
converted into a Uracil (both CpG and C)
– Remember you only have the Sense strand of DNA (you
must account for the anti-sense)
– Your PCR product should be between 100-300 bp
– All primers (S and AS) are written 5’ to 3’
– Please write down sequence to primers as well as
underlining on the sequence
CpG island to design MSP primers from:
•
•
•
•
•
•
•
•
•
•
Written 5’ to 3’ (remember this is only written single stranded)
1 CGGGTCCCACCTCGCAGGCCAGCTGGAGGGCGCGATCCTGGCGTCCCCCG
53 ACGGCCTGGGGCCCCAATCCAGAGGCCTGGGTGGGAGGGGACCAAGGGT
102 GTAGTAAGGAAGCGCCTTTTGCTGGAGGGCAACGGACCGGGGCGGGGAGTC
153 GGGAGACCAGAGTGGGAGGAAGGCGGGGAGTCCAGGTTCCGCCCCGGAGCC
204 GACTTCCTCCTGGTCGGCGGCTGCAGCGGGGTGAGCGGCGGCAGCGGCCGGG
256 GATCCTGGAGCCATGGGGCGCGCGCGCGACGCCATCCTGGATGCGCTGGA
306 GAACCTGACCGCCGAGGAGCTCAAGAAGTTCAAGCTGAAGCTGCTGTCGGTG
358 CCGCTGCGCGAGGGCTACGGGCGCATCCCGCGGGGCGCGCTGCTGTCCATGGA
411 CGCCTTGGACCTCACCGACAAGCTGGTCAGCTTCTACCTGGAGACCTACGGC
Comparing Methylation and Expression Data
•
1.
After analyzing the MSP and reverse transcriptase PCR from
breast cancer cells lines analyze the following questions
Do methylation and gene expression correlate? If so, in what
way?
2.
Why can’t we just do PCR directly from RNA?
3.
Why is ß-actin shown for each sample?
4.
Does this data support or refute your conclusions from part I?
MSP for gene X
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Reverse Transcriptase PCR for gene X
Analyzing bisulfite sequencing results
• Draw the lollipop figure of DNA methylation status for the
following 2 sequences:
Original sequence: 5’ACGTCGCGTCCACCGCGTAAGGTCGCTCGACGATCGTCG 3’
Sequence of clone #1:
5’ATGTTGTGTTTATCGCGTAAGGTTGTTTGACGATCGTCG 3’
Sequence of clone #2:
5’ ACGTCGCGTTTATCGCGTAAGGTCGTTCGACGATCGTCG 3’
Follow up questions to bisulfite sequencing analysis
1.
Which clone #1 or #2 would you hypothesize came from the tumor and which
from the normal tissue? Why?
2.
If you had an unknown tissue sample and you wanted to know the
methylation status of your favorite gene. What technique would you use and
why?
3.
Which technique to you think would be most suited to look at the effects of
epigenetic therapy (DNA methylation inhibitors) on a tumor sample? Why?