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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

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DNA SEQUENCING

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  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

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