biotechniques. dna sequencing to determine the base sequence of dna fragments. dna replicated into...
TRANSCRIPT
Biotechniques
DNA sequencingTo determine the base sequence of DNA fragments.
DNA replicated into smaller fragments incorporating fluorescent tags
Fragments separated by gel electrophoresis.
DNA sequence is determined from the order of the fluorescent coloured bands.
Fluorescent tags are on analogue bases A*, G*, C* and T*
Prepare purified DNA Sample
Create Tagged FragmentsPCR machine multiplies DNA sample
Uses primers and nucleotides (normal and tagged)
Fluorescent tags analogue bases A*, G*, C* and T*
Making DNA fragmentsDNA replicated - mixture of normal and tagged nucleotides.
As soon as a tagged nucleotide is added to the growing chain, replication stops.
normal tagged
lots of fragments of different length, the end of the fragments all tagged with fluorescent dye
lots of fragments of different length, the end of the fragments all tagged with fluorescent dye
Fra
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nRunning the Gel
Large fragments travel slowly
Gel through which the DNA fragments travel
Small fragments travel quickly
T G G C A T A C T A A Yellow
T G G C A T A C T A Green
T G G C A T A C T Green
T G G C A T Green
T G G C Green
T G G C A T A Blue
T G G C A T A C Red
T G G C A Red
T G G Blue
YellowT G
Red
T Yellow
A
G C
T
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G C
TT G G C A T A C T A A Yellow
T G G C A T A C T A Green
T G G C A T A C T Green
T G G C A T Green
T G G C Green
T G G C A T A Blue
T G G C A T A C Red
T G G C A Red
T G G Blue
YellowT G
Red
T Yellow Yellow tag = G
Red tag = T
Green tag = A
Blue tag = C
Yellow tag = G
T GG C A T A C T AA GDNA sequence
shortest sequence = 1 nucleotide
Controlled by powerful computer software
The gel is left to run for up to 10 hours.
Running the DNA Sequencer
Analyzing the DataData from the digital
camera is collected by computer software.
Each tagged base is given a different color on the graph.
DNA sequencing Videos
• http://www.youtube.com/watch?v=lVA89P2mfYk&feature=related (DNA seq., 6 min – better)
• http://www.youtube.com/watch?v=xgbVG2Nx6Ko&feature=related (DNA seq., 3 min – not simple)
• DNA SEQUENCING is an example of a technique that uses other techniques!