define euchtomatic sequence
genomes that are easy to transcribe
what is the human genome project
international endeavour that resulted in the sequencing of the human genome to 99.99% accuracy
how long did the human genome project take
1990- april 2003
cost of genome project then vs now
$100 mil to $1000 USD
what type of sequencing did the human genome start with
sanger sequencing!
what type of technology performs DNA sequencing
Polymerase Chain Reaction (PCR)
why is DNA called DEOXYribonucleic acid?
DNA lacks the -OH group on C2
where is the phosphodiester bond located
between C5 of the 5’ nucleotide and C3 of the 3’ nucleotide- mediated by DNA polymerase
what do dideoxynucleotides lack?
ddNTPs lack the -OH group on C2 and C3
what does removing the OH group do?
it means no phosphodiester bond which means elongation stops
other names for sanger sequencing
-chain termination
-dideoxy sequencing method
key steps of sanger sequencing
- primer made to known sequence
- shear template into smaller fragments
- sequence reaction using low concentration of ddNTPs
- newly synthesized DNA terminated by the random incorporation of ddNTPs
- DNA separated by size using gel electropheresis
(small fragments first)
how many genes at once in sanger sequencing?
one
describe whole genome sequencing process
notes**
describe targeted panel sequencing
- 40 to 400 genes
- high coverage
- search bias
describe whole exome sequencing
-22 000 genes
- intermediate coverage
- only sequencing exomes for certain gene
describe whole genome sequencing
- lower coverage
- all genes, translocations and non coding DNA
- pro: cover whole genome
- con: no deep coverage per gene for higher accuracy
define coverage
how many times a DNA region is sequenced
how do DNA microarrays work
-DNA to mRNA to protein through gene expression
-mRNA to cDNA through reverse transcription
what type of genes are transcribed into mRNA
protein coding genes
what does it mean if cDNA hybridizes
the gene is expressed
what is whole exome sequencing (WES)
- protein coding regions are captured and isolated using DNA microarray techniques
- non coding regions are washed away prior to sequencing
- sequencing is only performed on coding DNA
what is first generation sequencing
- sanger sequencing
- one gene/time
- DNA fragments: 500-1000 bp; amplification required
- time to sequence whole genome is 10 years
- expensive
- highly accurate
define 2nd generation sequencing
- next generation sequencing
- massively parallel sequencing
- DNA fragments: 100-500 bp; amplification required
- time to sequence genome= 1 week
- less accurate than sanger sequencing due to coverage
