DNA Sequensing

DNA Sequencing

DNA sequencingDetermination of nucleotide sequence

the determination of the precise sequence of nucleotides in a sample of DNA

Two similar methods:1. Maxam and Gilbert method

2. Sanger method

They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in

length by a single nucleotide at the other end This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands

determined

Maxam-Gilbert• Walter Gilbert

– Harvard physicist– Knew James Watson– Became intrigued with

the biological side– Became a biophysicist

• Allan Maxam

Sanger MethodFred Sanger, 1958

– Was originally a protein chemist

– Made his first mark in sequencing proteins

– Made his second mark in sequencing RNA

1980 dideoxy sequencing

Original Sanger MethodRandom incorporation of a dideoxynucleoside

triphosphate into a growing strand of DNARequires DNA polymerase I

Requires a cloning vector with initial primer (M13, high yield bacteriophage, modified by adding: beta-

galactosidase screening, polylinker)Uses 32P-deoxynucleoside triphosphates

Sanger Method in-vitro DNA synthesis using ‘terminators’, use of dideoxi-

nucleotides that do not permit chain elongation after their integration

DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific nucleotides

Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system

Incorporation of di-deoxynucleotides into growing strand terminates synthesis

Synthesized strand sizes are determined for each di-deoxynucleotide by using gel or capillary electrophoresis

Enzymatic methods

Dideoxynucleotide

no hydroxyl group at 3’ endprevents strand extension

CH2O

OPPP5’

3’

BASE

The principles• Partial copies of DNA fragments made with DNA

polymerase• Collection of DNA fragments that terminate with

A,C,G or T using ddNTP• Separate by gel electrophoresis

• Read DNA sequence

CCGTAC3’ 5’5’ 3’primer

dNTP

ddATPGGCA

ddTTP

GGCAT

ddCTP

GGC G

ddGTP

GGGGCATG

A T C G

DideoxyChain Terminator

TemplatePrimer

Extension Chemistry– polymerase– termination

– labelingSeparationDetection

Chain Terminator Basics

TargetTemplate-Primer

ExtendddA

ddG

ddC

ddTLabeled Terminators

ddA

AddC

ACddG

ACG ddT

TGCA

dN : ddN100 : 1

Electrophoresis

Sanger Method Sequencing Gel

Template ssDNA vectors

– M13– pUC

PCRdsDNA (+/- PCR)

PrimersUniversal primers

– cheap, reliable, easy, fast, parallelparallel– BULK sequencing

Custom primers– expensive, slow, one-at-a-time

– ADAPTABLEADAPTABLE

ExtensionPolymerase

– Sequenase– Thermostable (Cycle Sequencing)

Terminators– Dye labels (“Big Dye”)

• spectrally different, high fluorescence– ddA,C,G,T with primer labels

SeparationGel ElectrophoresisCapillary Electrophoresis

– suited to automation• rapid (2 hrs vs 12 hrs)• re-usable• simple temperature control• 96 well format

Sample Output

1 lane

Sequencing of DNA by the Sanger method

Apa yang harus disequense??

• 16S rDNA –mitokondria• Sitokrom b• Mikrosatelit

endosymbiosis:endosymbiosis:

• origin of nucleus pre-dates endosymbiosis

• ancestors of eukaryotes lacking mitochondria either never underwent endosymbiosis, or did and organelle subsequently lost

evolutionary chronometer: a molecule whose sequence can be used as a comparative measure of evolutionary divergence

small subunit ribosomal RNA (ssu rRNA)small subunit ribosomal RNA (ssu rRNA)

universal phylogenetic tree (tree of life):universal phylogenetic tree (tree of life):

genomic hybridization:genomic hybridization:• possible to use as a taxonomic tool

(i) label DNA of one test organism (ii) denature DNA of all organisms

genomic hybridization:genomic hybridization:

(iii) conduct hybridization experiment by mixing DNA from pairs of organisms

• unlabelled DNA added in excess(iv) separate hybridized from unhybridized DNA

• measure radioactivity in hybridized

genomic hybridization:genomic hybridization:

(iv) assess results• amount of label (radioactivity) in control taken

as 100% hybridization

usual criteria:• hybridization = 70% or above → same species• needs to be at least 20-30% to argue that 2 isolates are

from same genus

ranges of DNA base composition:ranges of DNA base composition: