lecture 3- sequencing human genomes Flashcards
DNA replication
DNA double helix replicates itself by synthesizing copies of each strand
PCR
uses pair of flanking primers to synthesis many copies of the region of DNA that lies in between
Recombinant DNA
DNA can be copied and stuck together with other bits
Chain termination (SANGER) DNA sequencing
synthesis of many labelled copies of DNA of different lengths and separating by size
aligning sequencing
sequences of DNA that overlap can be aligned using computers and assembled into longer fragments/contigs
Sanger sequencing steps technique
- Many copies of the DNA are made
- Each primer is extended by DNA Polymerase until a dideoxynucleotide is incorporated
- DNA fragments of different sizes are generated
- DNA fragments are passed through a capillary to separate them by size. detector reads fluorescent tags
Shotgun sequencing strategy
- Smash it up- break up many DNA copies
- Add linkers (ligate pieces of known sequences)
- Sequence using primers that bind to linkers
- Assemble reads overlapping into contigs
genome sequencing challenges
sequenced several times, variation is retained
time cost
confusing parts can be resolved by reference genome
Why so few human genes
size of genome not indicative of number of genes
many genes turned out pseudogenes defective
how do we find genes ORFs
stretches of dna code begin ATG start codon
computer searches:TATA box, GC box, ORFs, splice sites
final exon ends with a stop codon
how do we find genes: Transcriptome
Make a DNA copy of all mRNA within cell/tissue
get representation of the transcriptome genes
Expressed sequence tags (ESTs)
how do we find genes: Comparative genomics
line up sequences of related genomic DNA and look at highly conserved similar regions (synteny)
more similarity= likely gene
