Exam 3 Part 4 Flashcards
Why sequence genomes?
understand genetic variation with respect to phenotypic variation
inheritance
comparison to other organism’s genomes
forensics
undertand genetics of extinct species
give insight into norml functions of genes
pharmacogenomics
Whole genome shotgun approach
- Extract DNA from cells
- cut DNA into small, overlapping fragments using restriction enzymes (performed in suboptimal conditions so not all sites cut)
- clone contigs to make a genomic library
- sequenced each clone using Sanger sequencing
- used computers to reassemble sequences of contigs by puzzling together overlapping sequences
- Deposited sequence information into NCBI bank
Sanger method
aka dideoxy sequencing, chain terminating
based on PCR of DNA template
If you don’t know sequence, how can you design a primer?
- can’t design a primer against an unknown sequence
2. can use a universal primer and the same one
contigs
DNA fragments of various sizes (overlapping)
ddNTP
dideoxynucleic acid
Only has H instead of OH on 3’ so phosphodiester bond cannot continue adding and synthesis stops
After reaction is complete
an array of products with flourescent molecules attached are separated by size using capillary gel electrophoresis
reassembling sequence
repeat sequencing for each clone in library
reassemble contigs using overlapping sequences
What we have learned
sequences of other organisms 3.2 billion baspairs about 20,000 protein coding genes 5,000 genes do not code for protein introns are large genome is only 2% genes average gene is 3,000 bp genes are clustered together on chromosomes 99.9% sequence in common with other people
largest gene
dytrophin -2.4 million bp
What we haven’t learned
long stretches of repeated DNA sequence that wre hard to reassemble
genes vs pseudogenes vs dubious ORFs
what some gene products actually do
How we find protein coding genes vs all other sequences
compare cDNA library to genomic library
use computer algorithms to look for consensus sequences
annotation
identification and description of genes and their important sequences
goal to assign functions to all of the genes in an organism
used to understand variation within an among organisms
identify where traits come from
Next generation sequencing
extract DNA cut into contigs affix DNA to solid support one by one, wash DNA with dNTPS If the known dNTP is incorporated, light emmits reassemble using overlapping sequences
DNA marker
a specific region of DNA that varies among individuals
used to create a detailed map of the individual’s genome
