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7/21/2019 DNA Sequencing_Blok 9

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DNA SequencingHow do you do it?


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What & Why?

Sequencingmeans fnding the ordero nucleotides on a piece o DNA .

Nucleotide order determines Amino acid

order and !y e"tension proteinstructure and unction #proteomics$

An alteration in a DNA sequence canlead to an altered or non unctional

protein and hence to a harmul e%ect ina plant or animal


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What & Why td.

'nderstanding a particular DNAsequence can shed light on a geneticcondition and o%er hope or the e(entual

de(elopment o treatment DNA technology is also e"tended to

en(ironmental agricultural and orensicapplications


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DNA Sequence (ariation can change the )roteiproduced !y a particular gene

Simple point mutations such as this can causealtered protein shape and unction.

Diseases such as Sic*le ell Anaemia and ys+i!rosis are caused !y point mutations

,A A,A ,A- AA- -,-,ene Aromperson

odon results inparticular AminoAcid #AA$ sequence

Ala Arg Asp Asn ys

,, A,A ,A- AA- -,-,ene Aromperson /

odon changema*es no dierencein AA#redundant code$

Ala Arg Asp Asn ys

,A AAA ,A- AA- -,-,ene Aromperson 0

odon changeresults in di%erentAA sequence

Ala 1ys Asp Asn ys


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Historically there are two mainmethods o DNA sequencing2

3a"am & ,il!ert usingchemical sequencingSanger using

dideoxynucleotides.3odern sequencing equipmentuses the principles o the Sanger

technique.


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-he Sanger -echnique

'ses dideo"ynucleotides#dideo"yadenine dideo"yguanine etc$

-hese are molecules that resem!lenormal nucleotides !ut lac* the normal

45H group.


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Nucleoside (dNT)

Nucleotide (dNTP)


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6ecause they lac* the 45H #which allowsnucleotides to 7oin a growing DNAstrand$ replication stops.

Normally, this wouldbe where anotherphosphateIs attached, but withno -OH

group, a bond can notform and replication

stops


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-he Sanger method requires

3ultiple copies o single strandedtemplate DNA

A suita!le primer#a small piece o DNAthat can pair with the template DNA toact as a starting point or replication$

DNA polymerase#an en8yme that copiesDNA adding new nucleotides to the 09end o the template

A :pool9 o normal nucleotides

A small proportion o dideo"ynucleotidesla!eled in some way # radioacti(ely orwith ;uorescent dyes$


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PCR Reagents

1X Buffer 10mM Tris-HCl, 50mM KCl

MCl! 1mM - "mM (1#5mM)

dNTPs !00$M

Primers 100%M-1$M, !00%m (or less) for re&l time &%&l'sis

N *ol'mer&se TaqN *ol'mer&se is t+ermost&le 1-" %its (1 u%it)

N (tem*l&te N) 10*-1$ (!0%)

+

ddNTPs


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-he template DNA pieces are replicatedincorporating normal nucleotides !ut

occasionally and at randomdideo"y#DD$ nucleotides are ta*en up.

-his stops replication on that piece oDNA

-he result is a mi" o DNA lengths eachending with a particular la!eledDDnucleotide.

6ecause the di%erent lengths :tra(el9 atdi%erent rates during electrophoresistheir order can !e determined.

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