DNA SEQUENCING
Dr. Edwin Ginés-
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
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)
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
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
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
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http://web.virginia.edu/Heidi/chapter12/chp12.htm
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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
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
Hydrazine dilute buffer
Hydrazine in 2 M NaCl
Piperidine
Piperidine
– 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
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
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
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MAXAM-GILBERT CHEMICAL CLEAVAGE
PROCEDURE
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MAXAM-GILBERT SEQUENCING GEL RESULTS
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