watsgoingon
Badges: 6
Rep:
?
#1
Report Thread starter 7 years ago
#1
hi there,

http://care.diabetesjournals.org/con...4/386.full.pdf

from this article, can someone please explain to me about the genomic DNA. was it ever possible to clone the insulin from genomic DNA? many thanks!!
0
reply
Rob da Mop
Badges: 15
Rep:
?
#2
Report 7 years ago
#2
In figure 3 and the section titled "cloning the insulin gene from genomic DNA" they show how the human insulin gene can be isolated and cloned for study. Does that answer your question, or is there something else about it that confuses you? Also, I assume you've been given this paper to read - if you found it by yourself you do realise it's 40 years old, right?
0
reply
watsgoingon
Badges: 6
Rep:
?
#3
Report Thread starter 7 years ago
#3
(Original post by Rob da Mop)
In figure 3 and the section titled "cloning the insulin gene from genomic DNA" they show how the human insulin gene can be isolated and cloned for study. Does that answer your question, or is there something else about it that confuses you? Also, I assume you've been given this paper to read - if you found it by yourself you do realise it's 40 years old, right?
i couldn't understand because i was just reading through the gene cloning but it doesn't say that complementary DNA (cDNA) is produced from mature mRNA before incorporating into the vectors.all it says that DNA is cut by restriction endonuclease and inserted in to vectors. i have to write an essay about cloning the insulin gene and i am very confused. i am searching from other sources but i am not able to find anything.
0
reply
Rob da Mop
Badges: 15
Rep:
?
#4
Report 7 years ago
#4
(Original post by watsgoingon)
i couldn't understand because i was just reading through the gene cloning but it doesn't say that complementary DNA (cDNA) is produced from mature mRNA before incorporating into the vectors.all it says that DNA is cut by restriction endonuclease and inserted in to vectors. i have to write an essay about cloning the insulin gene and i am very confused. i am searching from other sources but i am not able to find anything.
cDNA is used in the section I think we're discussing as a probe to identify which bacteria contain the insulin gene. cDNA is NOT inserted into the vectors. The vectors have DNA unnecessary for their life removed, and replaced with random sections of human GENOMIC DNA, not cDNA. These then infect bacteria. Once these bacteria are cultured the ones containing the fragment of DNA with the insulin gene can be identified by using a radioactive (32-P) probe of the cDNA for insulin. When this hybridises with insulin mRNA or related sequences in the bacteria they can be identified and then further cultured, allowing us to identify the sequence as it exists in human DNA. I hope that helps.

Not really sure what else I can do to help -if you have any specific questions then quote me but my brain's too frazzled atm for me to try to read that whole paper right now.
1
reply
watsgoingon
Badges: 6
Rep:
?
#5
Report Thread starter 7 years ago
#5
(Original post by Rob da Mop)
cDNA is used in the section I think we're discussing as a probe to identify which bacteria contain the insulin gene. cDNA is NOT inserted into the vectors. The vectors have DNA unnecessary for their life removed, and replaced with random sections of human GENOMIC DNA, not cDNA. These then infect bacteria. Once these bacteria are cultured the ones containing the fragment of DNA with the insulin gene can be identified by using a radioactive (32-P) probe of the cDNA for insulin. When this hybridises with insulin mRNA or related sequences in the bacteria they can be identified and then further cultured, allowing us to identify the sequence as it exists in human DNA. I hope that helps.

Not really sure what else I can do to help -if you have any specific questions then quote me but my brain's too frazzled atm for me to try to read that whole paper right now.

thank you for replying. but it says in here in point number 5, its just a few quotes from somewhere:
sorry about the length :-(

How is this recombinant insulin gene cloned?
1. RNA that codes for insulin is extracted from pancreatic cells.
2. A complimentary strand of DNA (cDNA) is built onto the RNA using the enzyme reverse transcriptase.
3. Enzymes split the cDNA & RNA strands.
4. The addition of nucleotides & DNA polymerase to the single cDNA strand build a 2nd strand on it. This DNA has special linking sequences built onto each end so it will fit into the plasmid the right way round.
5. The DNA is inserted into a plasmid which also contains a gene for resistance to an antibiotic. To do this a restriction enzyme cuts open the plasmid (the “scissors”), the cDNA is inserted & DNA ligase “glues” the 2 different DNA’s together. This recombinant plasmid now contains a human gene.
6. The plasmid is mixed in with E. coli & is transfected into the bacteria.
7. Each plasmid replicates, making many copies inside its bacterium (i.e. cloning the gene).
8. The bacteria are put into the antibiotic – only those with the insulin DNA inserted into their plasmid will survive. This is how the transfected bacteria are separated from bacteria without the recombinant DNA in their plasmids.
9. The bacterial culture goes into a fermentation tank where the bacteria (& their plasmids) reproduce. With this recombinant plasmid DNA E. coli can process the instructions to assemble the amino acids for insulin production & they make lots. The trade name for insulin produced from GE E. coli is Humulin. Yeast is also used (trade name = Novolin).
0
reply
Rob da Mop
Badges: 15
Rep:
?
#6
Report 7 years ago
#6
Where have you got those points from? I assume "How is this recombinant insulin gene cloned?" is the question you're trying to answer. That list already looks like a good short bullet-point answer. Sorry if I'm being useless I'm just struggling a bit to see what you want help with!
0
reply
X

Quick Reply

Attached files
Write a reply...
Reply
new posts
Back
to top
Latest
My Feed

See more of what you like on
The Student Room

You can personalise what you see on TSR. Tell us a little about yourself to get started.

Personalise

Are you tempted to change your firm university choice on A-level results day?

Yes, I'll try and go to a uni higher up the league tables (49)
28.99%
Yes, there is a uni that I prefer and I'll fit in better (14)
8.28%
No I am happy with my choice (94)
55.62%
I'm using Clearing when I have my exam results (12)
7.1%

Watched Threads

View All
Latest
My Feed