Lecture 26 Flashcards
How many bases do humans have?
about 3.2Gb
How many genes do humans have?
About 21,000 genes
What is the Sanger method? How is it done?
“Sequence by synthesis”.
- DNA separated into 2 strands.
- Strand to sequence is copied using chemically altered bases or PCR.
- Each base encountered causes growing DNA chain to stop (depending on which base it stops at) and to terminate - will be different lengths. This is done for all 4 bases and then larger fragments put together like a jigsaw.
What does the sanger method need in order to occur?
Template DNA Sequence primer Taq polymerase AGCT (deoxy nucleotides) 4 ddNTP's (labels) at low concentration
Describe capillary electrophoresis.
Small scale, fast, high output.
How much does the Sanger method ‘read’?
1Kb per read.
What is pyrosequencing?
Seeing a flash of light each time a base is added. It is cheap, fast and automatable.
How is light produced in the pyrosequencing method?
If CTP is added, Sulfurylase takes PPi -> ATP. Luciferase takes ATP -> Light. Also apyrase.
What is the homo-polymer error?
In pyrosequencing and ion torrent, if 2 bases added, you get twice the response. 3 bases are added, you get triple the response, etc. When it gets to 9, error applies.
What is the Ion Torrent method of sequencing?
There is a proton given off when nucleotide added which decreases the pH. There will be twice the change in pH when 2 bases are added, etc, thus homo-polymer error applies. It is a fast mechanism.
What is an ORF?
An open reading frame. There are 6 reading frames in DNA (3 forward, 3 back). An ORF is the one with no stop codons. This is how genes are identified.
What is the expected frequency of a stop codon in a random sequence?
1/64 bases.
