Landsberger: Lecture XVIII

Question 1

Why can using a restriction enzyme technique be useful?

Answer 1

it can help identify whether the genome is methylated or not, but it cannot detect sequences

Question 2

Why is using restriction enzymes with radioactive probes useful?

Answer 2

this can detect if a gene or gene promoter is methylated

Question 3

What is a southern blot?

Answer 3

technique used to understand if a specific fragment of DNA has a different MW after hybridization

Question 4

What did the use of restriction enzymes prove?

Answer 4

there is a difference in the methylation of ribosomal DNA between vertebrates (more methylations) and intervertebrates

Question 5

What are the limitations with using restriction enzymes with radioactive probe?

Answer 5

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

Question 6

What was a technique developed to replace restriction enzymes with radioactive probe?

Answer 6

restriction enzymes with PCR

Question 7

If the DNA is unmethylated and I perform PCR, _____.

Answer 7

the 200 bp fragment is not detected

Question 8

If the DNA is methylated and I perform PCR, _____.

Answer 8

PCR amplification will occur

Question 9

What are the limitation with using restriction enzymes with PCR?

Answer 9

it is limited by restriction enzymes

if DNA is not clean, it will be amplified undigested because PCR is incredibly sensitive

Question 10

What technique solves the problem of undigested DNA being amplified when using unclean DNA with restriction enzymes and PCR?

Answer 10

quantitative PCR

Question 11

What is a technique that checks if CG nt are methylated or not?

Answer 11

Bisulfite treatment (followed by PCR, qPCR, microarrays, DNA sequencing)

Question 12

Sodium bisulfite works only on _____.

Answer 12

single strand DNA

Question 13

How does the sodium bisulfite technique work?

Answer 13

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

Question 14

What are the limitations to the Bisulfite technique?

Answer 14

denature is very strong…could destroy DNA

methyl cytosines and hydroxymethyl cytosines remain

Question 15

What technique should I use if I want to check if I have created or destroyed restriction sites?

Answer 15

using combined disulfide restriction analysis (COBRA)

Question 16

What is the easiest technique to use if I know I have a disorder?

Answer 16

bisufite treatment followed by methylation sensitive PCR

Question 17

How if bisulfite treatment followed by methylation sensitive PCR run?

Answer 17

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)

Question 18

What is MRE-Seq?

Answer 18

technique using the idea that we are going to select restriction enzymes (3-5) that are methylation sensitive

Question 19

What are some limitations to MRE-Seq?

Answer 19

expensive and coverage is limited to 30% of our genome

Question 20

What is MeDIP Seq?

Answer 20

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

Question 21

What are the limitations to MeDIP Seq?

Answer 21

we still won’t know how many cytosines are methylated and in which position

Question 22

What is methyl binding domain (MBD seq)?

Answer 22

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)

Question 23

What is a limitation of MBD Seq?

Answer 23

it does not tell you where methylation is occuring

Question 24

What was learned from combining MeDIP and MRE-Seq?

Answer 24

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

Question 25

How does transcription work in prokaryotic cells?

Answer 25

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

Question 26

In prokaryotic cells, most transcription is ____.

Answer 26

basal

Question 27

Why do we (eukaryotes) use methylation?

Answer 27

we need it for chromatin compaction

Question 28

If a gene is a houskeeping gene (=always expressed), _______.

Answer 28

it does not have a lot of methylation

Question 29

DNA methylation →

Answer 29

silencing (no transcription)

Question 30

No DNA methylation →

Answer 30

transcription

Question 31

Methyltransferase I is :

Answer 31

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

Question 32

What is interesting in regards to methylation with the zygote?

Answer 32

it is quite methylated

Question 33

What happens after the first division of the zygote?

Answer 33

there is a wave loss of DNA methylation

Question 34

Which genome is methylated more quickly?

Answer 34

paternal genome (sperm)

Question 35

Describe the pattern of methylation in somatic cells?

Answer 35

it is quite stable; big changes occur during development

Question 36

What is currently thought about DNA methylation?

Answer 36

in somatic cells, CGs are methylated

Question 37

Do neurons gain or lose methylations?

Answer 37

neurons gain methylations

Question 38

In neural tumors, there is a ___ of DNA methylation.

Answer 38

loss

Question 39

Why is methylation important?

Answer 39

protects the integrity of our DNA