About MMLV RT

[kokoioi]

Main Question or Discussion Point

how MMLV RT can end labeling of DNA?It is a RNA-dependent DNA polymerase,but i read some paper use it to label dna which after Restrict enzyme digestion?? expect your reply.thx

 

[Monique]

MMLV RT: Moloney Murine Leukemia Virus Reverse Transcriptase

You might find the following technical literature from Epicentre usefull:
http://www.epicentre.com/pdftechlit/050pl092.pdf [Broken]

They summarize some of the different applications of MMLV RT:

- Synthesis of cDNA
- mRNA 5’-end Mapping by Primer Extension Analysis
- RT-PCR
- Dideoxynucleotide Sequencing
- End-labeling of DNA
- Synthesis of Radioactive cDNA Probes

For each application they have a reference to literature, for the End-Labeling of DNA they reference to:
4. Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed.) New York: Cold Spring Harbor Laboratory Press.

Epicentre: "MMLV RT can be used to radioactively label DNA fragments with a 5'-protruding termini in a fill-in reaction using the appropriate radioactive dNTP(s)."

 

[Monique]

So to answer your question more fully:

If your restriction digest results in 5'-protruding termini, you can use MMLV RT to fill in the ends with radioactively labeled dNTPs (you label those with T4 polynucleotide kinase).

I just wonder, can't other enzymes not be used? Like Klenow?

 

[kokoioi]

hi~Monique

thx for your reply.
It is mean MMLV RT can use DNA for template to synthesis new strand?

[zz)]

 

[Monique]

Originally posted by kokoioi
hi~Monique

thx for your reply.
It is mean MMLV RT can use DNA for template to synthesis new strand?

[zz)]

Yes :) I think it needs to be double stranded though..

 

[kokoioi]

hi~~i think Klenow also can use to label.

i read a paper in our seminar.
molecular microbiology.(48)p901-911
"Sequence-specific recognition but position-dependent cleavage of two distinct telomeres by the Borrelia burgdorferi telomere resolvase,ResT"

I worte a mail to Mr.Chasconas(auteur).why use MMLV RT?
and auteur told me "This enzyme is used as a polymerase to add the [a-32P] C to the Xba cut DNA (it is added across from the G in the sticky end). "

and the cleavagesite of XbaI
5'-T^C T A G A-3'
3'-A G A T C^T-5'
(the experiment is for location )
so i think MMLV RT may dont primer when it use DNA for template.
what do you think? and i can't find about MMLV RT use to label DNA in my college.

 

[iansmith]

There migth be some explanation there

http://www.epicentre.com/f3_3rt.asp [Broken]
http://www.epicentre.com/pdftechlit/050pl092.pdf [Broken]

 

[Monique]

Originally posted by kokoioi
and the cleavagesite of XbaI
5'-T^C T A G A-3'
3'-A G A T C^T-5'
(the experiment is for location )
so i think MMLV RT may dont primer when it use DNA for template.
what do you think? and i can't find about MMLV RT use to label DNA in my college.

I am not entirely sure of your question. Are you asking whether MMLV RT needs a primer to extend the DNA?

If so, the answer is yes and no. The primer is already there after your restriction digest. Remember that after digestion your DNA will look like this:

5'- C T A G A N N N N -3'
3'-         T N N N N -5'

The MMLV RT will fill in the 5' overhang with labeled nucleotides. Just make sure that you DO NOT denature your restriction digest!!! Maybe that is why MMLV RT is used, because it works at a low temperature?