Current Level

 Replication
 Transcription
 Translation
 PCR
 DNA synthesis
 DNA sequencing

Previous Level

 BioWeb Home
 
 Seq Anal
 Theory
 Bioinformatics
 Mo Bio Lecture
 
 DNA sequencing

DNA sequencing reactions all use a primer to initiate DNA synthesis.  This primer will determine the starting point of the sequence being read, and the direction of the sequencing reaction.

Most DNA sequencing reactions use dideoxy nucleotides (ddNTP) to stop DNA synthesis at specific nucleotides.  For example, if the ddCTP to the right is incorportated into a growing strand of DNA, the lack of a free 3 OH group would prevent the next nucleotide from being added, and the chain would terminate.

In automated sequencing we use a different fluorescent label attached to each of the four dideoxy nucleotides (ddA, ddC, ddG and ddT).  Thus we can determine the terminal base in each fragment of DNA. 

The two animations below illustrate how DNA sythesis and dideoxy termination are used to sequence DNA.

Dc

Deoxycytosine (dCTP)

Ddn

Dideoxycytosine (ddCTP)

 


ANIMATIONS

A Quicktime animation of how dideoxy nucleotides are incorporated into DNA in sequencing reactions.

videoicon1

videoicon1

A Shockwave animation of dideoxy sequencing from Cold Spring Harbor.  http://www.dnalc.org/ddnalc/resources/sangerseq.html

A Shockwave animation of automated thermocycle seqeuncing from Cold Spring Harbor.     http://www.dnalc.org/ddnalc/resources/cycseq.html

    

DNA SEQUENCING GELS

Examples of gels from automated sequencing (left) and manual sequencing (right). 

Automated sequencing uses a different fluorescent dye attached to each ddNTP. 

Manual sequencing uses radiolabeled dATP (35-S or 33-P) to label the DNA.  The sample is then split into four tubes  each with an individual ddNTP present.  The samples are then subjected to acrylamide gel electrophoresis followed by autoradiography.

Click on either gel to go to the sequence analysis site.

DNAgel

Automated Sequencing

courtesy of UW-Biotech Center

uwsa_l5

  2002 The Board of Regents of the University of Wisconsin System.

Click here to email comments to Scott Cooper regarding this site or its links.

Gel

   G      A      T    C

Manual Sequencing