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

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

8. Fragments travel in this direction Running 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 Yellow T G Red T Yellow A GC T

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

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

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 http://www.youtube.com/watch?v=lVA89P2mfYk &feature=related (DNA seq., 6 min – better) http://www.youtube.com/watch?v=lVA89P2mfYk &feature=related http://www.youtube.com/watch?v=xgbVG2Nx6K o&feature=related (DNA seq., 3 min – not simple) http://www.youtube.com/watch?v=xgbVG2Nx6K o&feature=related DNA SEQUENCING is an example of a technique that uses other techniques!