1 / 20

DNA SEQUENCING

DNA SEQUENCING. Dr. Edwin Gin és-Candelaria. DETERMINING THE BASE SEQUENCE OF DNA. MAXAM-GILBERT PROCEDURE Basis ssDNA derived from dsDNA is subjected to chemical treatments that cleave DNA molecules —> family of short ssDNA fragments

beverly-leblanc
Download Presentation

DNA SEQUENCING

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. DNA SEQUENCING Dr. Edwin Ginés-Candelaria

  2. DETERMINING THE BASE SEQUENCE OF DNA • MAXAM-GILBERT PROCEDURE • Basis • ssDNA derived from dsDNA is subjected to chemical treatments that cleave DNA molecules —> family of short ssDNA fragments • No. nucleotides per fragment determined by gel electrophoresis • PAGE separates DNA molecules differing by a single nucleotide • Several cleavage protocols used; each is base specific and provides the sequence following the rationale: • If a fragment contains n nucleotides and is generated by treatment active against a particular base; then that base is in position n + 1of the DNA strand, the position being counted from the 5’ end

  3. THE MAXAM-GILBERT PROCEDURE • BASIS • If a 36 bp fragment results from the protocol that ID guanine (G), then it is known that G is in the 37th base in the original molecule • RESOLUTION • Up to 300 nucleotides • Fragment obtained by digestion w/ 1 or more restriction enzymes • Fragments (32P ) end labeled 5’ end w/ polynucleotide kinase • Radiolabeled DNA denatured by treatment w/ NaOH • Labeled ssDNA fragments separated by electrophoresis (each strand is sequenced separately & confirm one another)

  4. THE MAXAM-GILBERT PROCEDURE • PROCEDURE • Sample containing the purified ssDNAs is divided into 2 portions Portion I - add dimethylsulfate that methylates Purines G is methylated 5X more effectively than A (partial rxn not carried to completion ensures that only one purine/ssDNA is methylated)-b/c methylation occurs randomly, the particular A or G methylated differs in each strand Methylated DNA sample is divided into 2 portions Ia, Ib

  5. THE MAXAM-GILBERT PROCEDURE • PROCEDURE • Portion Ia heated • Heat treatment removes all methylated bases • Alkaline treatment (cleaves sugar-phosphate backbone @ the site where the base has been removed • Heat/alkaline cleavage protocol treatment —> set of fragments of differing in size & no. nucleotides. The latter is determined by different positions of methylated G or A • B/c G is methylated >>> A, sample Ia is said to contain G-only fragments

  6. THE MAXAM-GILBERT PROCEDURE • PROCEDURE • Portion Ib diluted acid • Removes all methylated A, but some G & called A + G fragments; note in the gel that every G-only fragment size is present in the A + G collection • Two samples Ia & Ib —> electrophoresed in 20% PAGE in presence of 8 M urea (denaturing conditions) • Autoradiography locates the bands on the exposed film (several day exposure) • Single terminal 32P is the sole source of radioactivity for detection

  7. http://www.nd.edu/~aseriann/maxgilchem.html

  8. http://web.virginia.edu/Heidi/chapter12/chp12.htm

  9. http://web.virginia.edu/Heidi/chapter12/chp12.htm

  10. THE MAXAM-GILBERT PROCEDURE • PROCEDURE • Positions of A & G in the ssDNA - determined by the following rules: • If a band containing n nucleotides is present in both A + G and the G-only lane, the G exists at position n + 1 in the original molecule • If a band containing n nucleotides is present only in the A + G lanes, then A exists at position n + 1 in the original molecule

  11. THE MAXAM-GILBERT PROCEDURE • PROCEDURE • Analysis of Sample II - use to ID the positions of C & T • Sample II divided into two portions IIa & Iib • IIa IIb • C + T C-only fragments • Hydrazine reacts w/ C and T (but not A nor G), but in 2 M NaCl, it reacts w/ C only • After electrophoresis and autoradiography Hydrazine dilute buffer Hydrazine in 2 M NaCl Piperidine Piperidine

  12. THE MAXAM-GILBERT PROCEDURE • PROCEDURE • Positions of C & T in the ssDNA - determined by the following rules: • If a band containing n nucleotides is present in both C + T and the T-only lane, the C exists at position n + 1 in the original molecule • If a band containing n nucleotides is present only in the C + T lanes, then T exists at position n + 1 in the original molecule • All 4 samples (Ia, Ib, Iia, Iib) are electrophoresed simultaneously so all bands are observed in a single gel • Sequence read directly from gel w/ shortest fragments moving the farthest & containing the original 5’-32P • Sequence is then read from bottom to top

  13. THE MAXAM-GILBERT PROCEDURE • PROCEDURE • Note that where a band in the autoradiogram is labeled w/ base X, this means that X was removed from the 3’ end of the DNA molecule to generate the fragment • Thus, if only one ssDNA of the original dsDNA molecule were analyzed, then the complete sequence would not be obtained. This is due to: • If cleavage is at position n + 1 (counting from 32P-end labeled terminus), the no. of bases in the fragment is n, which means that, no fragment IDs the 5’-terminal nucleotide • Mononucleotide that would ID the penultimate base can’t be detected unless we sequence the complementary strand and ID the 3’ terminal bases • B/c resolution is limited to 300 nucleotides, then sets of overlapping fragments must be obtained and sequenced

  14. http://biology200.gsu.edu/houghton/4564%20%2704/lecture4.htmlhttp://biology200.gsu.edu/houghton/4564%20%2704/lecture4.html

  15. http://homepages.strath.ac.uk/~dfs99109/BB211/MGSeq.html

  16. MAXAM-GILBERT CHEMICAL CLEAVAGE PROCEDURE http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html

  17. MAXAM-GILBERT SEQUENCING GEL RESULTS http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html http://nationaldiagnostics.com/article_info.php/articles_id/20

More Related