DNA Sequencing - Lecture notes

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Molecular Biology (MTY1210)

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Far Eastern University

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DNA SEQUENCING - refers to sequencing methods for determining the order of the nucleotide bases (adenine, guanine, cytosine, and thymine) MAXAM-GILBERT METHOD (1970, ALLAN M. MAXAM & WALTER GILBERT) – later SANGER METHOD (originally developed by FREDERICK SANGER, 1975) DNA – template used to create series of fragments (fragments differ by 1 base, separated by SIZE) CHROMOSOME – cut down into smaller fragments to make it feasible MANUAL DNA SEQUENCING – most specific and direct method for identifying genetic lesions/ mutations/ polymorphisms (affecting 1 or 2 nucleotides) (Types: SANGER METHOD / MAXAM-GILBERT METHOD) MAXAM-GILBER METHOD – chemical sequencing, requires a double- or single-stranded version of the DNA region to be sequenced with one end radioactively labeled (aliquoted into 4 tubes; with or w/o salt) 10% PIPERIDINE – strong reducing agent, breaks single- stranded DNA at specific nucleotides Tube 1 – DIMETHYLSULPHATE (breaks at G) Tube 2 – FORMIC ACID (breaks at G + A) Tube 3 – HYDRAZINE (breaks at T + C) Tube 4 – HYDRAZINE + SALT (breaks at C) BOTTOM (5’) to TOP (3’) – how sequence is read G + A and G = GUANINE G + A = ADENINE C + T and C = CYTOSINE C + T = THYMINE HYDRAZINE & PIPERIDINE – hazardous chemicals SANGER METHOD (DIDEOXY CHAIN TERMINATION SEQUENCING) – ( 19 77, FREDERICK SANGER & COLLEAGUES) widely used sequencing methods for 25 years after its discovery, required a single-stranded template. It is a modification of the DNA replication process. M13 BACTERIOPHAGE - a bacterial virus with a single stranded DNA genome DIDEOXYNUCLEOTIDE (ddNTP) - lack the hydroxyl group found on the 3′ ribose carbon of the deoxynucleotides (dNTPs). Newly synthesized chain will terminate with the ddNTP AUTOMATED FLUORESCENT SEQUENCING - uses double-stranded templates and cycle sequencing (cycle sequencing is more easily adaptable to high throughput applications and automation) Goal of both approaches is the same: to label the fragments synthesized during the sequencing reaction according to their terminal ddNTP DYE PRIMER SEQUENCING - Four different fluorescent dyes are attached to four separate aliquots of the prime. Primer labeled with each “color” is added to four separate reaction tubes, one each with ddATP, ddCTP, ddGTP, or ddTTP The products of the sequencing reaction are then labeled at the 5′ end, the dye color associated with the ddNTP at the end of the fragment DYE TERMINATOR SEQUENCING - performed with one of the 4 fluorescent dyes attached to each of the ddNTPs instead of to the primer. Primer is unlabeled All four sequencing reactions are performed in the same tube (or well of a plate) instead of in four separate tubes

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DNA SEQUENCING - refers to sequencing methods for

determining the order of the nucleotide bases (adenine,

guanine, cytosine, and thymine)

MAXAM-GILBERT METHOD (1970, ALLAN M. MAXAM

& WALTER GILBERT) – later SANGER METHOD

(originally developed by FREDERICK SANGER, 1975)

DNA – template used to create series of fragments

(fragments differ by 1 base, separated by SIZE)

CHROMOSOME – cut down into smaller fragments to

make it feasible

MANUAL DNA SEQUENCING – most specific and direct

method for identifying genetic lesions/ mutations/

polymorphisms (affecting 1 or 2 nucleotides) (Types:

SANGER METHOD / MAXAM-GILBERT METHOD)

MAXAM-GILBER METHOD – chemical sequencing,

requires a double- or single-stranded version of the DNA

region to be sequenced with one end radioactively

labeled (aliquoted into 4 tubes; with or w/o salt)

10% PIPERIDINE – strong reducing agent, breaks single-

stranded DNA at specific nucleotides

Tube 1 – DIMETHYLSULPHATE (breaks at G)

Tube 2 – FORMIC ACID (breaks at G + A)

Tube 3 – HYDRAZINE (breaks at T + C)

Tube 4 – HYDRAZINE + SALT (breaks at C)

BOTTOM (5’) to TOP (3’) – how sequence is read

G + A and G = GUANINE

G + A = ADENINE

C + T and C = CYTOSINE

C + T = THYMINE

HYDRAZINE & PIPERIDINE – hazardous chemicals

SANGER METHOD (DIDEOXY CHAIN TERMINATION

SEQUENCING) – (1977, FREDERICK SANGER &

COLLEAGUES) widely used sequencing methods for 25

years after its discovery, required a single-stranded

template. It is a modification of the DNA replication

process.

M13 BACTERIOPHAGE - a bacterial virus with a single

stranded DNA genome

DIDEOXYNUCLEOTIDE (ddNTP) - lack the hydroxyl group

found on the 3′ ribose carbon of the deoxynucleotides

(dNTPs). Newly synthesized chain will terminate with

the ddNTP

AUTOMATED FLUORESCENT SEQUENCING - uses

double-stranded templates and cycle sequencing (cycle

sequencing is more easily adaptable to high throughput

applications and automation)

Goal of both approaches is the same: to label the

fragments synthesized during the sequencing reaction

according to their terminal ddNTP

DYE PRIMER SEQUENCING - Four different fluorescent

dyes are attached to four separate aliquots of the prime.

Primer labeled with each “color” is added to four

separate reaction tubes, one each with ddATP, ddCTP,

ddGTP, or ddTTP

The products of the sequencing reaction are then

labeled at the 5′ end, the dye color associated with the

ddNTP at the end of the fragment

DYE TERMINATOR SEQUENCING - performed with one

of the 4 fluorescent dyes attached to each of the

ddNTPs instead of to the primer.

Primer is unlabeled

All four sequencing reactions are performed in the same

tube (or well of a plate) instead of in four separate tubes

DNA Sequencing - Lecture notes

Molecular Biology (MTY1210)

Far Eastern University

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