2. Genetics 7: Analyzing DNA Sequences

3. DNA Sequencing Determining base by base the nucleotide sequence of a fragment of DNA.

4. Manual Sequencing Dideoxy sequencing: a way to sequence the DNA that uses the principles of DNA replication. –DNA polymerase is used to made a series of fragments –The fragments all start at the same position, but they finish at a variety of different spots because they use a different nucleotide that cannot form any more bonds. These nucleotides are called dideoxynucleotides (ddA, ddG, ddC, ddT

5. Dideoxy Sequencing Steps DNA to be sequenced is denatured to a single strand and a primer anneals to the 3’ end Four separate reactions are made. They all contain the single stranded DNA to be sequenced, primers, regular nucleotides, DNA polymerase and one of the four dideoxynucleotides

6. Steps Continued… DNA synthesis reaction starts. Fragments of DNA is made in a few different lengths depending on where the dideoxynucleotide binds on. The four different reactions are placed in separate wells of a gel electrophoresis apparatus and is turned on. The shortest fragments travel the furthest through the gel, so scientists can “read” the gel by identifying the letters in order of shortest to longest.

7. Dideoxy Sequencing

8. Sequence this:

9. Early Automated DNA Sequencing Many scientists came together to complete the Human Genome Project where they sequenced the entire human genome. This is a huge amount of DNA and we needed a faster technique. Used dyed dideoxynucleotides so we don’t need the 4 separate reaction tubes and we can run them all on the same gel and use a photo-detector hooked up to a computer to read the sequence.

10. Recent Discoveries… Changing from dideoxy sequencing to “next-generation sequences” This constant attempt to improve and speed up the technique is important for: –Cancer diagnosis and treatments (detect point mutations, chromosomal rearrangements) Tumour profiling: sequence the DNA of cancerous cells Define types of cancer –Cheaper and faster

11. Making Sequence-Specific Mutations Scientists can now cause a mutation at a specific nucleotide and observe what would happen (site-directed mutagenesis) to the resulting protein.

12. Questions: Page 300 # 7, 8, 9, 11.