Exploring MMLV RT for DNA End Labeling: A Comprehensive Guide

  • Thread starter kokoioi
  • Start date
In summary, MMLV RT is a RNA-dependent DNA polymerase that can be used to label DNA after restriction enzyme digestion. It does not require a primer, as the overhang from the restriction digest serves as the template for the labeled nucleotides. It can also work at a lower temperature, which may be beneficial for this specific application.
  • #1
kokoioi
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 :smile:
 
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  • #2
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)."
 
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  • #3
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?
 
  • #4
hi~Monique

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

[zz)]:smile:
 
  • #5
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)]:smile:
Yes :) I think it needs to be double stranded though..
 
  • #6
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 don't 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.
 
  • #7
There migth be some explanation there

http://www.epicentre.com/f3_3rt.asp [Broken]
http://www.epicentre.com/pdftechlit/050pl092.pdf [Broken]
 
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  • #8
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 don't 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:

Code:
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?
 

1. What is MMLV RT and how is it used for DNA end labeling?

MMLV RT stands for Moloney Murine Leukemia Virus Reverse Transcriptase, which is an enzyme commonly used in molecular biology experiments to convert RNA into complementary DNA (cDNA). In DNA end labeling, MMLV RT is used to add a fluorescent or radioactive label to the end of a DNA molecule, allowing for easier detection and visualization.

2. What are the benefits of using MMLV RT for DNA end labeling?

MMLV RT is highly specific and can accurately label the ends of DNA molecules, making it a reliable method for studying DNA sequences. It is also relatively easy to use and does not require specialized equipment.

3. Can MMLV RT be used for labeling both single-stranded and double-stranded DNA?

Yes, MMLV RT can be used to label both single-stranded and double-stranded DNA. However, it is important to note that the labeling efficiency may vary depending on the type of DNA being labeled.

4. Are there any alternative methods for DNA end labeling?

Yes, there are alternative methods for DNA end labeling such as T4 DNA ligase or terminal deoxynucleotidyl transferase (TdT). These methods may be more suitable for certain applications, so it is important to consider the specific needs of the experiment before choosing a labeling method.

5. What are some potential troubleshooting tips for using MMLV RT for DNA end labeling?

If you are having trouble labeling your DNA using MMLV RT, some potential solutions include optimizing the reaction conditions (such as temperature and incubation time), using higher concentrations of the enzyme, or trying a different labeling method. It may also be helpful to consult with experienced researchers or refer to published protocols for guidance.

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