Landsberger: Lecture XVIII Flashcards
Techniques to Study Methylation
Why can using a restriction enzyme technique be useful?
it can help identify whether the genome is methylated or not, but it cannot detect sequences
Why is using restriction enzymes with radioactive probes useful?
this can detect if a gene or gene promoter is methylated
What is a southern blot?
technique used to understand if a specific fragment of DNA has a different MW after hybridization
What did the use of restriction enzymes prove?
there is a difference in the methylation of ribosomal DNA between vertebrates (more methylations) and intervertebrates
What are the limitations with using restriction enzymes with radioactive probe?
many restriction enzymes do not check if CG methylation occurs
most restriction enzymes do not have control (isoschizomer)
time consuming & uses radioactivity which is expensive and not sensitive
What was a technique developed to replace restriction enzymes with radioactive probe?
restriction enzymes with PCR
If the DNA is unmethylated and I perform PCR, _____.
the 200 bp fragment is not detected
If the DNA is methylated and I perform PCR, _____.
PCR amplification will occur
What are the limitation with using restriction enzymes with PCR?
it is limited by restriction enzymes
if DNA is not clean, it will be amplified undigested because PCR is incredibly sensitive
What technique solves the problem of undigested DNA being amplified when using unclean DNA with restriction enzymes and PCR?
quantitative PCR
What is a technique that checks if CG nt are methylated or not?
Bisulfite treatment (followed by PCR, qPCR, microarrays, DNA sequencing)
Sodium bisulfite works only on _____.
single strand DNA
How does the sodium bisulfite technique work?
DNA is incubated in harsh conditions to be fragmented
DNA is denatured
DNA is then incubated with sodium disulfite, which turns non-methylated cytosines into uracil
remaining C’s are methylated
What are the limitations to the Bisulfite technique?
denature is very strong…could destroy DNA
methyl cytosines and hydroxymethyl cytosines remain
What technique should I use if I want to check if I have created or destroyed restriction sites?
using combined disulfide restriction analysis (COBRA)
What is the easiest technique to use if I know I have a disorder?
bisufite treatment followed by methylation sensitive PCR
How if bisulfite treatment followed by methylation sensitive PCR run?
the bisulfite steps are the same as before; however in PCR, we will use 2 primers (one that amplifies the methylated DNA and the other will amplify the unmethylated DNA)
What is MRE-Seq?
technique using the idea that we are going to select restriction enzymes (3-5) that are methylation sensitive
What are some limitations to MRE-Seq?
expensive and coverage is limited to 30% of our genome
What is MeDIP Seq?
a technique that takes advantage of a monoclonal antibody generated against methylcytosine
after using the bisulfite technique, we can used this to distinguish between methylated and hydroxymethylated
What are the limitations to MeDIP Seq?
we still won’t know how many cytosines are methylated and in which position
What is methyl binding domain (MBD seq)?
uses the methyl-DNA binding domain of the reader with high affinity for highly methylated DNA
MBD binds to methylated DNA and the concentration of CL determines how many fragments of methyl groups are released (ex: if we increase CL, there will be a medium level of methylation)
What is a limitation of MBD Seq?
it does not tell you where methylation is occuring
What was learned from combining MeDIP and MRE-Seq?
unicellular eukaryotes do not have DNA methylation (no DNA methyltransferase)
C. elegans does not have DNA methylation (no DNA methyltransferase)
invertebrates have an intermediate level of methylation
vertebrates have a high level of methylation
plants are more methylated than we are
How does transcription work in prokaryotic cells?
https://courses.lumenlearning.com/wm-biology1/chapter/prokaryotic-transcription/
https://www.youtube.com/watch?v=WsofH466lqk&embeds_euri=https%3A%2F%2Fcourses.lumenlearning.com%2F&feature=emb_logo
In prokaryotic cells, most transcription is ____.
basal
Why do we (eukaryotes) use methylation?
we need it for chromatin compaction
If a gene is a houskeeping gene (=always expressed), _______.
it does not have a lot of methylation
DNA methylation →
silencing (no transcription)
No DNA methylation →
transcription
Methyltransferase I is :
an important enzyme for DNA methylation
cannot be the only enzyme because if it were, we would have 100% methylation
ESCs can survive and replicate without DNA methylation
What is interesting in regards to methylation with the zygote?
it is quite methylated
What happens after the first division of the zygote?
there is a wave loss of DNA methylation
Which genome is methylated more quickly?
paternal genome (sperm)
Describe the pattern of methylation in somatic cells?
it is quite stable; big changes occur during development
What is currently thought about DNA methylation?
in somatic cells, CGs are methylated
Do neurons gain or lose methylations?
neurons gain methylations
In neural tumors, there is a ___ of DNA methylation.
loss
Why is methylation important?
protects the integrity of our DNA
