BIOL 142 Study Guide - Final Guide: Point Mutation, Growth Factor, Threonine
9 May 2018
School
Department
Course
Professor
BIO142 Levy
Exam 3 Review
1
BIO$142:$Foundations$of$Modern$Biology$II!
Midterm$Exam$3$Review$
!
Genomics$I$
!
Learning!Objectives:!
1. Define!genomics!
2. Describe!the!use!of!dideoxynucleotide!triphosphates!(ddNTPs)!in!DNA!sequencing!reactions!
3. Explain!the!7!steps!of!shotgun!sequencing!
4. Explain!how!open!reading!frames!(ORF)!and!expressed!sequence!tags!(EST)!are!used!to!
identify!functional!genes!
!
1.!Define!genomics!
Genomics:!the!scientific!effort!to!sequence,!interpret,!and!compare!whole!genomes;!provides!a!list!of!
the!genes!present!in!an!organism!
• Genome:!the!complete!DNA!sequence!of!an!organism!
• Functional!genomics:!looks!at!when!those!genes!are!expressed!and!how!their!products!interact!
o Expression!information!–!when!and!where!are!genes!being!turned!on!a!making!products!
• Proteomics:!study!of!proteins!and!how!they!work!in!a!large!scale!
!
2.!Describe!the!use!of!dideoxynucleotide!triphosphates!(ddNTPs)!in!DNA!sequencing!reactions!
Is!based!on!an!in#vitro#DNA!synthesis!reaction!and!requires!many!of!the!same!components!used!in!a!PCR!
reaction,!including!
• DNA!Template!is!sequenced!
• Deoxyribonucleotides!(dNTPs)!are!needed!as!building!blocks!for!the!DNA!
o Normal!dNTPS!are!included!to!produce!a!range!of!DNA!synthesis!products!that!
terminate!at!every!occurrence!of!a!particular!base!(not!just!get!information!about!one!
position)!
• DNA!Polymerase!
• DNA!Primers!provides!3'!ends!for!the!DNA!polymerase!to!incorporate!bases!on!
• Dideoxynucleotide!triphosphates!(ddNTPs)!are!also!used!in!addition!to!the!use!of!dNTPs!in!the!
synthesis!reactions!
o How!this!differs!from!PCR!
!
Dideoxynucleotide!triphosphates!(ddNTPs):!chain-elongating!inhibitors!of!DNA!polymerase,!used!in!the!
Sanger!method!for!DNA!sequencing!
• Are!identical!to!dNTPs!except!that!they!lack!the!3'!hydroxyl!group!
• When!a!dideoxyribonucleotide!triphosphate!(ddNTP)!is!incorporated!into!the!3’!end!of!a!
growing!DNA!strand!by!DNA!polymerase!ddNTPs!stop!DNA!polymerization!because!they!lack!a!
hydroxyl!(-OH)!group!at!their!3ʹ!end!
BIO142 Levy
Exam 3 Review
2
• DNA!polymerization!stops!once!a!ddNTP!is!added!to!a!growing!strain!
• Each!ddNTP!has!a!distinct!fluorescent!label!for!each!base!(A,!G,!C,!T)!
o Ends!up!in!results!as!a!series!of!peaks!
o Peaks!show!confidence!à!higher!=!more!confident!because!=!more!fluorescence!=!
higher!concentration!of!DNA!fragments!
!
3.!Explain!the!7!steps!of!shotgun!sequencing!
Shotgun!sequencing:!consists!of!breaking!open!the!genome!into!many!small!fragments,!sequencing!
each!fragment,!and!then!putting!the!sequence!data!back!in!the!correct!order!via!computer;!used!when!
researchers!first!set!out!to!sequence!the!genome!of!a!species!
• Needed!because!DNA!polymerase!can!only!extend!so!far!
o Can’t!just!use!2!primers!to!sequence!a!whole!chromosome!!
o Sanger!sequence!primers!are!too!limited!
• In!the!shotgun!genome!sequencing!approach,!160!kb!genomic!DNA!fragments!are!broken!into!1!
kb!fragments!(in!“shotgun!clones”)!that!are!sequenced!because!are!too!long!to!sequence!with!
Sanger!!
o Need!smaller!fragments!and!lots!of!breakage!so!you!can!get!pieces!of!overlap!
• Since!the!breaks!are!random!and!there!are!many!copies!of!the!genomic!DNA,!these!fragments!
are!expected!to!overlap!in!many!regions!
!
Steps!of!shotgun!sequencing!
1. Cut!DNA!at!random!locations!into!fragments!~160!kb!–!Application!of!high-frequency!sound!
waves,!or!sonication,!is!used!to!break!a!genome!randomly!into!pieces!about!160-kb!long!
2. Clone!using!BACs!–!Each!160-kb!piece!is!inserted!into!an!engineered!version!of!a!bacterial!
chromosome!called!a!bacterial!artificial!chromosome!(BAC),!that!can!be!used!as!a!cloning!
vector.!!
o BACs!are!able!to!replicate!large!segments!of!DNA!
o Each!BAC!is!then!inserted!into!a!different!bacterial!cell!
o By!allowing!each!cell!to!grow!into!a!colony,!researchers!can!isolate!large!numbers!of!
each!160-kb!fragment.!
3. Cut!into!1!kb!fragments!–!After!many!copies!of!each!160-kb!fragment!have!been!produced,!each!
cloned!DNA!is!broken!into!fragments!again,!but!this!time!about!1-kb!long!
4. Clone!using!plasmids!–!These!small!fragments!are!then!inserted!into!plasmids!and!placed!inside!
bacterial!cells.!The!plasmids!are!copied!many!times!as!each!cell!grows!into!a!large!population.!
5. Sequence!each!fragment!–!The!cloned!1-kb!fragments!from!each!160-kb!BAC!clone!are!
sequenced,!and!computer!programs!analyze!regions!where!the!ends!of!different!1-kb!fragments!
overlap.!
o Overlap!occurs!because!many!copies!of!each!160-kb!segment!were!made!and!then!
fragmented!randomly!by!sonication!
6. Assemble!all!the!1!kb!fragments!–!Based!on!the!overlap!between!1-kb!fragments!from!a!single!
BAC!clone,!the!computer!stitches!together!until!a!continuous!sequence!across!the!BAC!has!been!
reconstructed!
7. Assemble!all!the!160!kb!fragments!–!the!ends!of!the!reconstructed!BACs!are!analyzed!
o The!goal!is!to!link!sequences!from!each!160-kb!segment!on!regions!of!overlap!until!the!
entire!genome!is!assembled!!
BIO142 Levy
Exam 3 Review
3
!
!
!
!
!
4.!Explain!how!open!reading!frames!(ORF)!and!expressed!sequence!tags!(EST)!are!used!to!identify!
functional!genes!
Open!reading!frames!(ORFs):!long!stretches!of!sequence!that!lack!a!stop!codon!but!are!flanked!by!a!
start!codon!and!a!stop!codon;!potential!genes!based!on!characteristic!of!being!long!and!not!interrupted!
by!stops;!used!in!prokaryotes;!can’t!be!used!in!eukaryotes!because!of!introns!
• If!converted!into!an!RNA!sequence!and!translated!as!a!set!of!non-overlapping!codons,!would!
produce!a!polypeptide!of!substantial!size!
• Discovery!of!one!is!an!important!piece!of!evidence!to!identify!a!protein-coding!gene!
o Because!randomly!generated!sequences!contain!a!stop!codon!at!about!1!in!every!20!
codons!on!average,!the!presence!of!a!long!stretch!of!codons!that!lacks!a!stop!codon!is!a!
good!indication!of!a!protein-coding!sequence!
• Computer!programs!are!used!to!scan!a!prokaryotic!genome!sequence!in!both!directions!in!order!
to!identify!ORFs!
• 6!possible!reading!frames!in!a!double!stranded!DNA!molecule!
o First!reading!frame!incorporates!first!codon,!then!the!following!codons!are!read!
§ 5'!ATGCTAGCGC!-!3'!
o Can!also!ignore!the!first!base!and!start!the!codon!one!over!
§ 5'!ATGCTAGCGC!-!3'!
Document Summary
Genomics: the scientific effort to sequence, interpret, and compare whole genomes; provides a list of the genes present in an organism: genome: the complete dna sequence of an organism. Dideoxynucleotide triphosphates (ddntps): chain-elongating inhibitors of dna polymerase, used in the. In the shotgun genome sequencing approach, 160 kb genomic dna fragments are broken into 1 kb fragments (in shotgun clones ) that are sequenced because are too long to sequence with. Sanger: need smaller fragments and lots of breakage so you can get pieces of overlap. Since the breaks are random and there are many copies of the genomic dna, these fragments are expected to overlap in many regions. Levy: explain how open reading frames (orf) and expressed sequence tags (est) are used to identify functional genes. 5" atgctagcgc - 3: can also ignore the first base and start the codon one over. Exam 3 review: can ignore first 2 bases.
