What is the difference in between the Maxam-Gilbert Method and the Sanger Method?

I need the actual process

The samger method uses a DNA polymerase to synthesize a new DNA strand using a template and a specific primer, in the presence of dideoxynucleotides that terminate the synthesis specifically following the addition of an A, C, G, or T. In the modern incarnation, each dideoxy nucleotide is attached to a fluorescecnt dye so all of the fragments ending in a given base fluoresce the same color. Separating the fluorescent bands allows one to read of the color of each fragment that differ from each other by one nucleotide, and thereby read off the sequence.

The Maxam-Gilbert method takes a DNA fragment that has been labeled at the 5'-end, usually with radioactive phosphorus, and subjects to chemical cleavage with reagents that cut at specific bases. By using a low concenrtation of the reagents, only one or two sites are cleaved on each molecule. The products produced by cleavage are separated by electrophoresis and the bands made with the different reagents are run side by side and compared. Since only fragments with the labeled 5'-end are visible, the size of the fragment says that a site sensitive to that reagent (a specific base) is located x nucleotides from the 5'-end, again allowing the seqience to be read off.

Why does Sanger's method of sequencing allows all reactions to run on the same lane of gel?

My bio teacher didn't really explain it well.

It doesn't if you use radioactively labeled dideoxynucleotides. In order to run all the reactions in the same gel lane, you would have to use fluorescently labeled dideoxynucleotides. The Sanger or dideoxy chain-termination method uses dideoxynucleotides (ddATP, ddTTP, ddGTP, ddCTP), some normal nucleotides and DNA polymerase to extend the template DNA strand until a dideoxynucleotide is incorporated into the growing strand. When a dideoxynucleotide is incorporated the growth of the chain terminates. Each of the dideoxynucleotides is labeled, either with radioactivity (32P or 35S) or with a fluorescent tag. If you use a radioactive label, then each chain termination reaction must be run in a separate lane of the gel (because all of the bands will be detected with X-ray film and all will be black bands). If you use fluorescent tags, then each is a different color, so you can tell the different nucleotides apart and thus you can run them in the same lane if you want. This technique is used with automated sequencing equipment. Here is a diagram and further explanation of the flurorescent tag method:

http://users.rcn.com/jkimball.ma.ultran ... ncing.html

The Maxam-Gilbert method of DNA sequencing uses radioactively labeled DNA and chemical reactions that break the DNA chain at specific locations, allowing the sequence to be determined. Since radioactively labeled DNA is used, you must run each reaction in a separate lane of the gel in order to read the sequence. Here is a further explanation of Maxam-Gilbert sequencing:

http://www.nd.edu/~aseriann/maxam.html

The function of the dideoxy (dd) nucleotides that are used in the Sanger method of DNA sequencing is to ?

A. denature DNA into single strands.
B. serve as primers.
C. be a fluorescent tag.
D. incorporate into newly replicated DNA strands and stop elongation.
E. None of the choices are correct

D. incorporate into newly replicated DNA strands and stop elongation.

The dideoxy nucleotides are chain terminators because they lack an -OH group at the #3 postion of the ribose. This -OH group is what the next nucleotide needs to attach to in order to build onto the growing strand. So once a dideoxy nucleotide is incorporated, elongation stops because the next nucleotide has nothing to which it can attach.

In the actual Sanger sequence reaction mix most nucleotides are normal and only say like 1% are dideoxy. This allows for all possible lengths of the chain to be generated, and termination to occur randomly, thus building the ladder that you would see on a gel or fasimile of one.

How might RNA be sequenced indirectly using the Sanger squencing method?

How might RNA be sequenced indirectly using the Sanger squencing method?

Once you have your DNA dydeoxy readout, you go on to expasy and run a nucleotide comparison, then let the computer give you your rna readout.

OR!

If all you have is RNA, and RNA is really hard to 'sangerize', run a reverse transcription of your sample (reverse transcriptase... isn't it grand?!) and then run sanger dydeoxynucleotide sequencing on your newly formed dna... after a few pcr runs of course.

How does Fred Sanger's Dideoxy Method work?

In DNA sequencing?

The replication reaction requires the presence of a 3' OH on the last nucleotide inorder for the next nuclotide to be added. The reaction proceeds by nucleophilic attack of the 3' oxygen to the alpha phosphate of the incomming nucleoside triphosphate.

Sanger used this to advantage by realizing that if the 3' OH were removed then the replication reaction could be prematurly terminated.

Consider the following:

UNKNOWN SEQUENCE = ATTGCCA and its compliment

Reaction #1 contains ATP,GTP,CTP,TTP, and alpha- 32P-dideoxy ATP. Appropriate enzymes, primers and buffers.

The products of this reaction would be.

A
T

AT
TA

ATT
TAA

ATTGCCA
TAACGGT

You see that sometimes when an A is required the reaction stops because it incorporates the ddATP. Sometimes it continues because it incorporates dATP.

3 More reactions are required. In each one, the reaction is supplemented with a different ddNTP. These reactions stop differently than the ddATP reaction.

when these reactions are run sideby side on a gel you get a series of bands each one 1 nt longer than the next and stopping only in one of the reactions.

The sequence could thus be read examining the gel.

NOW a few other points
The base sugar in RNA is ribose
The base sugar in DNA is deoxyribose and lacks the 2' OH with several important biological implications.
Dideoxyribose lacks both the 2'OH and 3'OH and to my knowledge does not occur in nature.