BIOL 142 Chapter Notes - Chapter 20.3: Shotgun Sequencing, Reference Genome, Denisovan
30 Apr 2018
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20.3%DNA%Sequencing!
!!
DNA!Sequencing!
• Once!researchers!have!cloned!a!gene!or!amplified!a!region!of!DNA!by!PCR,!one!of!the!first!things!
they!usually!do!is!determining!the!DNA's!base!sequence!
• When!the!gene's!sequence!is!known,!the!genetic!code!can!be!used!to!infer!the!amino!acid!
sequence!of!the!gene's!protein!product!
o The!amino!acid!sequence!often!provides!clues!to!the!protein's!function!
• Comparing!sequences!is!fundamental!to!understanding!why!alleles!vary!in!function!
o Why!one!allele!causes!a!disease!and!another!doesn't!
• Evolutionary!relationships!can!be!inferred!by!comparing!the!sequences!of!the!same!gene!in!
different!species!
o Showed!Denisovans!to!be!a!new!human!lineage!
• Practical!methods!for!sequencing!DNA!date!back!to!1977!with!dideoxy!sequencing!
o Dideoxy(sequencing:!an!in!vitro!DNA!synthesis!reaction!technique!for!determining!the!
nucleotide!base!sequence!of!DNA;!relies!on!the!use!of!dideoxynucleoside!triphosphates!
(ddNTPs)!which!terminate!DNA!replication!
o Was!virtually!the!only!method!in!use!until!the!first!reports!of!the!human!genome!sequence!
in!2001!
o Still!performed,!usually!when!relatively!long!sequences!need!to!be!read!with!great!accuracy!
!!
Next-generation(sequencing:!a!number!of!different!DNA!sequencing!technologies!that!are!more!rapid!
and!cheaper!than!dideoxy!sequencing!
• Made!it!possible!to!rapidly!determine!the!sequences!of!entire!genomes!
• Now,!DNA!sequencing!technology!can!simultaneously!sequence!millions!of!DNA!fragments!
• Mostly!based!on!amplifying!steps!that!create!many!copies!of!each!template!DNA!molecule!
• Pros:!
o Allows!sequencing!of!minute!quantities!of!DNA!
o Millions!of!different!DNAs!can!be!amplified!and!sequenced!in!a!single!run!
o Faster!by!orders!of!magnitude!
• Cons:!
o They!produce!short!sequence!reads!of!only!about!50-200!nucleotides!
• Makes!it!challenging!to!piece!together!a!whole!genome,!especially!one!with!many!
repetitive!sequences!like!in!most!eukaryotes!
• Quick!and!inexpensive!way!to!determine!the!entire!genome!from!a!particular!genome!
• Arrange!all!the!tiny!fragments!in!the!correct!order!by!comparing!them!with!the!"reference!
genome"!
!!
Whole-Genome!Sequencing!
• A!single!dideoxy!sequencing!reaction!can!analyze!only!about!1000!nucleotides,!and!most!next-
generation!methods!give!much!shorter!read!lengths!
• Researchers!rely!on!shotgun!sequencing!!first!begin!to!sequence!the!genome!of!a!species!
o Shotgun(sequencing:!a!method!of!sequencing!genomes!that!is!based!on!breaking!the!
genome!into!small!pieces,!sequencing!each!piece!separately,!and!then!figuring!out!how!the!
pieces!are!connected!
• Many!copies!of!a!genome!are!broken!up!randomly!into!a!set!of!fragments!of!various!sizes!
Document Summary
Since the breaks are random and there are many copies of the genomic dna, these fragments are expected to overlap in many regions. Bioinformatics: the branch of computer science that is concerned with managing, analyzing, and interpreting biological information, particularly dna and amino acid sequences. For eukaryotes, sequencing data for protein-coding genes is more complicated: because coding regions are broken up by introns, it"s usually not possible to scan for long. Orfs: many approaches to find genes, can isolate mrnas from cells, use reverse transcriptase to produce a cdna version of a portion of mrna, and sequence the cdna to produce an expressed sequence tag (est) Expressed sequence tag (est): a cdna from a portion of a transcribed gene that is used to find the physical location of that gene in the genome and to confirm that a region of dna is transcribed.
