genetics 7: analyzing dna sequences dna sequencing determining base by base the nucleotide sequence...
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Genetics 7:
Analyzing DNA Sequences
DNA Sequencing
• Determining base by base the nucleotide sequence of a fragment of DNA.
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
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
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.
Dideoxy Sequencing
Sequence this:
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.
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
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.
Questions:
• Page 300 # 7, 8, 9, 11.
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