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.