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I'm trying to understand the method which Ray Wu, developed to sequence the DNA 5' cohesive ends of a lamda phage. I'm reading these two papers written by him, but i stuck at the point where he uses the 32P nucleotides.

Structure and base sequence in the cohesive ends of bacteriophage lambda DNA

Nucleotide sequence analysis of DNA: II. Complete nucleotide sequence of the cohesive ends of bacteriophage λ DNA

It's not clear to me, by reading the papers, the step in which he runs the polymerization of the DNA. Did he use all the 32P nucleotides at once or each one at a time. As i understood he made the DNA pol to use the tritiated nucleotides to fill in the gaps of the 5' cohesive ends and then he cleaves with a DNAase in many oligonucleotides which later fractionize with electrophoresis. If he added all tritiated nucleotides at once how did he recognize which one is at a specific place ?

Can someone describe the process in more detail using step by step aproach?

Thank you,

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  • $\begingroup$ Those two articles are located behind a paywall $\endgroup$ – RosieF Oct 23 '16 at 2:33
  • $\begingroup$ Do you want me, to upload them somewhere else or is it illegal? $\endgroup$ – F.N Oct 25 '16 at 8:53
  • $\begingroup$ I'm not a lawyer (I'm a biochemist). Don't worry, I think I have a new account for access. So you're still seeking an explanation? How did you come across these classics? $\endgroup$ – RosieF Oct 25 '16 at 12:11
  • $\begingroup$ I just wanted to see how people used to think back then with their only tool the analytical chemistry and some little knowledge of enzymes. 2 days i read it again and again and tried to put an order of the process/method he was used. His work is about 28 pages and he finally read about 20 nucleotides. Maxam's and Sanger's papers are around 8 pages and they cloud sequence 100 - 1000 nucleotides at once. Times are changing and it seems fascinating to me knowing a little bit of history. If you want the papers, let me know. $\endgroup$ – F.N Oct 25 '16 at 12:46
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    $\begingroup$ I am not going to post this as an answer, but I have skimmed the paper, and it is an absolute beast. Some facts worth noting. They do not use electrophoresis, they use ionophoresis. They also use a scintillation counter to calculate the number of molecules of radio labelled dNTP incorporated. After ionophoresis they purify the radioactive peaks and analyse those by nuclease digestion, and nearest neighbour analysis. Essentially they use the labels to purify the new oligos, and then fragment them to figure out the sequence of every dinucleotide. Then they build a map. $\endgroup$ – RosieF Oct 25 '16 at 17:02

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