Notices
Results 1 to 9 of 9
Like Tree2Likes
  • 1 Post By Skrith
  • 1 Post By Some

Thread: DNA Sequencing

  1. #1 DNA Sequencing 
    Forum Freshman
    Join Date
    Apr 2012
    Posts
    11
    DNA sequencing is a wonderful tool that we all have heard about. Although currently there are a lot of technologies that allow us to sequence DNA sequences, one of the first techniques to be used is called Sanger Sequencing.
    To perform a sequence analysis using the Sanger Sequencing technique it is necessary to pick primers to start DNA duplication. My question is: how do we know which primers to pick if we supposedly don't know the DNA sequence yet?


    Some likes this.
    Reply With Quote  
     

  2.  
     

  3. #2  
    Forum Freshman
    Join Date
    Dec 2012
    Location
    Farnham Royal, Bucks
    Posts
    65
    Presumably, in the good old days of amino acid sequencing, getting the amino acid sequence first and then guessing at the DNA sequence. Nowadays, and I left the field 20-odd years ago, so I am out of date, you would clone the DNA in a plasmid vector (there are a number of ways to do this). The sequence of the plasmid DNA is well known so that you can make primers complementary to the plasmid DNA and the primer is extended into the unknown sequence. You only need 20-30 base pairs of the unknown sequence and you can then make specific primers now for your 'unknown sequence'.

    This info is out of date so I am sure someone in the field will update it for you. But, it's a start...


    Reply With Quote  
     

  4. #3  
    Forum Freshman
    Join Date
    Apr 2012
    Posts
    11
    Yes, that's indeed a possibility. But I do know how we nowadays can (and I dare say easily) design the primers to such a sequencing analysis, even if we have no idea how the sequence we're analyzing is. But at the time Sanger devised this method they didn't know about the genetic code. They didn't know either the sequence of any vector (if I'm not mistaken, the first one to be sequenced was pBR322, and it was after Sanger and Gilbert developed their methods).
    Reply With Quote  
     

  5. #4  
    Forum Freshman
    Join Date
    May 2013
    Location
    West Virginia
    Posts
    32
    As I understand it, Sanger used the shotgun approach to sequence the bacteriophage and viral genomes initially. Chew them up and use them to randomly amplify the genome. The sequencing method itself, of course, does not rely on the use of primers. That's just to get enough fragments to sequence.

    After sequencing fragments, Sanger could identify the contiguous regions and put them back together. Sanger would have known about base pairing and the genetic code at the time, since it was known since the 50s and the 60s; his method was published in 1975. So that makes sense that he would have had an idea of what to look for and what a primer was all about.
    Care to read some more of what I have to say? Check out my blog!
    http://wwwscienceisforeveryone.net
    Reply With Quote  
     

  6. #5  
    Forum Professor Zwolver's Avatar
    Join Date
    May 2006
    Location
    Netherlands
    Posts
    1,676
    I think your overthinking it Skrith. You don't need a specific Primer. You only need a primer that gives the results you want. I agree with Deadally on the fact that he simply tested many different primers, and picked the one that gave medium length product, that contained the wanted fragment. You can check if you have the right fragment by using detection probes. A very time consuming, but to the core of biology interesting approach.
    Growing up, i marveled at star-trek's science, and ignored the perfect society. Now, i try to ignore their science, and marvel at the society.

    Imagine, being able to create matter out of thin air, and not coming up with using drones for boarding hostile ships. Or using drones to defend your own ship. Heck, using drones to block energy attacks, counterattack or for surveillance. Unless, of course, they are nano-machines in your blood, which is a billion times more complex..
    Reply With Quote  
     

  7. #6  
    Forum Freshman
    Join Date
    Apr 2012
    Posts
    11
    Yes, I believe I am. Thank you all for your answers!
    Reply With Quote  
     

  8. #7  
    Anti-Pseudoscience Some's Avatar
    Join Date
    Mar 2013
    Posts
    44
    This man is genius

    In 1977 Sanger and colleagues introduced the "dideoxy" chain-termination method for sequencing DNA molecules, also known as the "Sanger method".[15][17] This was a major breakthrough and allowed long stretches of DNA to be rapidly and accurately sequenced. It earned him his second Nobel prize in Chemistry in 1980, which he shared with Walter Gilbert and Paul Berg.[18] The new method was used by Sanger and colleagues to sequence human mitochondrial DNA (16,569 base pairs)[19] and bacteriophage λ (48,502 base pairs).[20] The dideoxy method was eventually used to sequence the entire human genome.
    He is thus far (2012) the only person to have been awarded two Nobel Prizes in Chemistry, and one of only four two-time Nobel laureates: the other three were Marie Curie (Physics, 1903 and Chemistry, 1911), Linus Pauling (Chemistry, 1954 and Peace, 1962) and John Bardeen (twice Physics, 1956 and 1972).
    Sanger sequencing - Wikipedia, the free encyclopedia


    Humanity have to thank him for his research. I admire him & his colleagues.
    SkyexSora likes this.
    Men are four: He who knows not and knows not he knows not, he is a fool--shun him; He who knows not and knows he knows not, he is simple--teach him; He who knows and knows not he knows, he is asleep--wake him; He who knows and knows he knows, hi is wise--follow him!
    Reply With Quote  
     

  9. #8  
    Forum Freshman
    Join Date
    May 2013
    Location
    West Virginia
    Posts
    32
    Some,

    For sure, his work is very, very important. It has taken until recently to really change the methods we're using now. With the advent of deep sequencing, Sanger's methods are finally being superceded (slowly, we still use a ton of Sanger sequencing)

    Your adoration gives me an idea for a blog post
    Care to read some more of what I have to say? Check out my blog!
    http://wwwscienceisforeveryone.net
    Reply With Quote  
     

  10. #9  
    New Member
    Join Date
    May 2014
    Posts
    1
    Mutations in DNA occur all the time. DNA sequencing methods, such as PCR or the Sanger method, use the replication of DNA to sequence it. How do scientists account for mutations that may (and probably do) occur during the process of sequencing?
    Reply With Quote  
     

Similar Threads

  1. Mouse Gut Microbe Sequencing
    By mafaisal in forum Biology
    Replies: 1
    Last Post: June 6th, 2011, 10:38 AM
  2. "Junk" DNA or Useful DNA?!?
    By forensicIRL2011 in forum Biology
    Replies: 1
    Last Post: March 5th, 2011, 01:40 PM
  3. Genetics - Nucleotide sequencing and using NCBI?
    By 0ochello0 in forum Biology
    Replies: 4
    Last Post: March 31st, 2009, 06:19 PM
  4. Dna polimerase types of DNA replication
    By SkyDreamer in forum Biology
    Replies: 2
    Last Post: March 2nd, 2008, 12:59 PM
  5. DNA
    By timmyg12 in forum Biology
    Replies: 3
    Last Post: December 17th, 2006, 05:04 PM
Bookmarks
Bookmarks
Posting Permissions
  • You may not post new threads
  • You may not post replies
  • You may not post attachments
  • You may not edit your posts
  •