About MMLV RT

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)."

 

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?

 

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

 

There migth be some explanation there

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

 

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:

Code (Text):

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?