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Biotechniques

Biotechniques. DNA sequencing. To 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.

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Biotechniques

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  1. Biotechniques

  2. DNA sequencing To 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*

  3. Prepare purified DNA Sample

  4. Create Tagged Fragments PCR machine multiplies DNA sample Uses primers and nucleotides (normal and tagged) Fluorescent tags  analogue bases A*, G*, C* and T*

  5. Making DNA fragments normal tagged As soon as a tagged nucleotide is added to the growing chain, replication stops. DNA replicated - mixture of normal and tagged nucleotides.

  6.  lots of fragments of different length, the end of the fragments all tagged with fluorescent dye

  7.  lots of fragments of different length, the end of the fragments all tagged with fluorescent dye

  8. Running the Gel T G G C A T A C T A A Yellow Green T G G C A T A C T T G G C A T A C Red T G G C A T A Blue T G G C A T Green T G G C A T A C T A Green T G G C A Red T G G C Green Blue T G G Yellow T G Yellow T Red Large fragments travel slowly Gel through which the DNA fragments travel G A T C Fragments travel in this direction Small fragments travel quickly

  9. T G G C A T A C T A A Yellow Green T G G C A T A C T T G G C A T A C Red T G G C A T A Blue T G G C A T Green T G G C A T A C T A Green T G G C A Red T G G C Green Blue T G G Yellow T G Yellow T Red T A C G T G G C A T A C T A A G Fragments travel in this direction DNA sequence Green tag = A Blue tag = C Yellow tag = G Yellow tag = G shortest sequence = 1 nucleotide Red tag = T

  10. Running the DNA Sequencer Controlled by powerful computer software The gel is left to run for up to 10 hours.

  11. Analyzing the Data • Data from the digital camera is collected by computer software. • Each tagged base is given a different color on the graph.

  12. DNA sequencing Videos • DNA SEQUENCING is an example of a technique that uses other techniques! 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)

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