genetics, cutting dna Best answer on the web

January 9, 2009 on 7:27 am | In mybachcars.com |
genetics, cutting dna Best answer on the web
  • how do enzymes cut dna at a specific point? and how do introns get spliced out (are the coding segments already gone in the mRNA sequences?)
    I have a science background, but in physics, not biology, so I can handle medium detail. I sometimes read something saying that enzymes are used to cut dna at a specific point, and wonder how it targets such a point, and cuts it.


  • Introns get cut out of pre-mRNAs by a class of enzymes called splieosomes. This splicing occurs before mRNAs are exported from the nucleus. You can see a simplistic video of a spliceosome working at http://www.neuro.wustl.edu/neuromuscular/pathol/diagrams/splicefunct.html. If you search for spliceosome on google you will get tons of hits.
    You can read about restriction enzymes (which cut DNA at specific locations; also called restriction endonucleases because they cut bonds between nucleotides in the middle of a strand of DNA rather than just the nucleotides at the end of a strand of DNA) at: http://www.accessexcellence.org/AE/AEC/CC/restriction.html or http://ghs.gresham.k12.or.us/science/ps/sci/ibbio/biotech/restriction.htm


  • Thanks for your comments, everyone.


  • okay here is a shot at explaining how restriction enzymes works at cutting specific sites of your gene.
    Your genes consist of a sequence of instructions made by arranging molecules in a polymer strand. The basic blocks used consist of a set of sugar-based molecules called Deoxyribonucleic Acid (DNA). DNA consist of a sugar unit-a phospate unit-and a "base" unit. There are 4 different kinds of "bases" in DNA: usually called Adenine (A), Guanine (G), Cytosine (C) and Thymidine (T). So your genes can be seen to consist of sequences containing different combination of AGCTGCTAGGCCTTAA.... and so on.
    Restriction enzymes were discovered from nature that acts on specific sequences i.e. this would mean specific points of your DNA/gene. Most of them will only cleave the gene/DNA where they encounter say a very specific sequence for e.g. an enzyme called AAT II will only cut when it sees the sequence 5"...GACGT^C...3" and cut at the point where the ^ sign is. By the way, your DNA is organized as a matched pair. So in reality there is an opposite set that mirrors the sequence, the 5" and 3" denotes the carbon positions of the sugar unit which forms the backbone of the DNA polymer. So in reality try and visualize the happening as follows:
    Original strand of DNA= 5"...GACGTC...3"
    3"...CTGCAG...5"
    Add AAT II
    you would get a cleave= 5"...GACGT3" + 5"C...3"
    3"...C5" 3"TGCAG...5"

    hope this helps a bit


  • First, the accessibility of genetic info depends on the ability of proteins to interact with DNA in a manner that allows it to be copied as DNA (replication) or RNA (transcription). You're asking about the transcription process. Now in prokaryotes, translation (protein synthesis) can begin even before transcription is done. Not so in eukaryotes. That first RNA that is transcribed is pre-mRNA and it will get some more modifications. Before it goes out to the cytosol (because it's made in the nucleus) it will have to get the intervening sequences (introns) cut out and the expressed sequences (exons) will get joined or spliced together. The introns have a high degree of homology among all of us eukaryotes-ie they typically have a GU at the 5' end and an AG at the 3' end. J. Steitz figured that one nucleic acid is probably best recognized by another and she found the small nuclear ribonucleproteins, snRNPs, or snurps. They are 60-300nt RNAs and their ends are partially complementary to the splice junction. Now the splicesome brings together the pre-mRNA and the snRNPs and a variety of other proteins to make the mature mRNA. The mechanism for proper splicing is still a mystery and exons can get cut out so that alternative splicing can give more than 1 protein product for a given gene. Oh boy, this got really long but I hope that's what your after.


  • I can't fully answer your question because I only know the basics myself, but here's a good primer. It has no mention of introns though, so *shrug* to that.
    http://library.thinkquest.org/19037/therapy2.html

    skermit-ga


  • limbatus, you gave me the key to the intron question, thanks! i'm starting to read through these links.
    by the first question, I mean, I sort of understand how DNA is (metaphorically) "read" by a ribosome when making protiens, in that free amino acids just match up with the nucleotides, so one thing codes the other in a sort of assembly line fasion.. but how does an enzyme "read" the nucleotide sequence to know where to cut?


  • Checkout New England Biolabs, http://www.neb.com/neb/tech/tech_resource/restriction/re_frame.html they have a good explanantion of restriction endonucleases, and a brief description of intron endonucleases, http://www.neb.com/neb/tech/tech_resource/restriction/intron_encoded/intron_frame.html


  • DNA is sectioned into small fragments by the use of enzymes. Enzymes are proteins that mediate reactions in a catalytic manner. The enzyme has an active site which binds to and reacts with specific nucleotide segments of the DNA.
    Specific enzymes react with, and bind to specific segments of DNA and thus, because of this specificity, different sections of the DNA molecule are hydrolyzed. The result is that the products of hydrolysis are of a different chemical composition and structure and thus, when assembled allow the total structure of the DNA molecule to re-created.