BIOL 142 Lecture 19: Genomics I
30 Apr 2018
School
Department
Course
Professor
4.##In#the#shotgun#genome#sequencing#approach,#160#kb#genomic#DNA#fragments#are#
broken#into#1#kb#fragments#(in#“shotgun#clones”)#that#are#sequenced.
Why#is#this#step#required#(ie—why#not#use#the#160#kb#genomic#DNA#fragments#as#
templates#for#sequencing#reactions)?
5. How#are#the#DNA#sequences#of#the#many#different#1#kb#fragments#in#shotgun#
clones#used#to#reconstruct#the#original#160#kb#genomic#DNA#fragments?
Overview
Define#genomics
•
Describe#the#use#of#dideoxynucleotide#triphosphates#(ddNTPs)#in#DNA#
sequencing#reactions
•
Explain#the#7#steps#of#shotgun#sequencing
•
Explain#how#open#reading#frames#(ORF)#and#expressed#sequence#tags#(EST)#are#
used#to#identify#functional#genes
•
Genomics:#the#scientific#effort#to#sequence,#interpret,#and#compare#whole#genomes
Genome:#the#complete#DNA#sequence#of#an#organism
The#human#genome#sequence#was#published#in#February#2001#as#part#of#
the#Human#Genome#Project
○
•
Genomics:#provides#a#list#of#the#genes#present#in#an#organism
•
Functional#genomics:#looks#at#when#those#genes#are#expressed#and#how#their#
products#interact
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
○
•
Which#Genomes#Are#Being#Sequenced,#and#Why?#
The#first#genome#of#an#organism#to#be#sequenced#was#that#of#the#bacterium#
Haemophilus,influenzae,in#1995#it#consists#of#about#1.8#million#base#pairs
Started#with#viruses#because#they#have#a#smaller#genome
○
•
The#first#eukaryotic#genome#to#be#sequenced#was#that#of#the#yeast#
Saccharomyces,cerevisiae,in#1996
Used#as#a#common#model#organism#and#for#commercial#use
Makes#bread#and#beer
§
○
Ferments#in#order#to#rise
○
•
To#date,#complete#genomes#have#been#sequenced#from#lots#of#species.#Most#of#
the#organisms#that#have#been#sequenced#cause#disease#or#have#other#
interesting#biological#properties
•
Dideoxy#DNA#sequencing#was#the#first#sequencing#method#developed#in#1975#by#
Fredrick#Sanger.#
The#method#is#based#on#an#in,vitro,DNA#synthesis#reaction#and#requires#many#of#
the#same#components#used#in#a#PCR#reaction,#including:#
DNA#Template#—something#to#sequenceA.
Deoxyribonucleotides#(dNTPs)#—synthesis#reaction,#need#these#as#
building#blocks#for#the#DNA
B.
DNA#PolymeraseC.
DNA#Primers—provides#3'#ends#for#the#DNA#polymerase#to#incorporate#
bases#on#the#end#of
D.
Dideoxynucleotide#triphosphates#(ddNTPs)#are#also#used#in#addition#to#
the#use#of#dNTPs#in#the#synthesis#reactions
How#this#differs#from#PCR
§
Stops#reaction#from#proceeding#any#further#because#it#lacks#OH#on#
the#C2#and#C3
§
E.
•
Dideoxynucleotide#triphosphates#(ddNTPs)#are#identical#to#dNTPs#except#that#they#
lack#the#3'#hydroxyl#group
dNTP#lacks#a#OH#on#the#C2,#just#has#a#hydrogen•
ddNTP#lacks#an#OH#on#the#C2#and#C3,#has#2#hydrogens•
2.#What#happens#when#a#dideoxyribonucleotide#triphosphate#(ddNTP)#is#incorporated#
into#the#3’#end#of#a#growing#DNA#strand#by#DNA#polymerase?#
ddNTPs#permanently#bind#to#the#active#site#of#DNA#polymerase.A.
ddNTPs#cause#DNA#polymerase#to#break#apart#into#small#fragments.#B.
ddNTPs#stop#DNA#polymerization#because#they#lack#a#hydroxyl#(-OH)#group#at#
their#3ʹ#end
Once#you#incorporate#a#ddNTP,#you#no#longer#can#add#any#more#
nucleotides
○
Nothing#for#DNA#polymerase#to#add#another#base#to
○
C.
ddNTPs#have#no#effect#upon#DNA#polymerization.D.
ddNTPs#cause#the#release#of#primers#from#the#template#DNA.#E.
Like#dNTPs,#ddNTPs#are#picked#up#by#DNA#polymerase#and#added#to#a#growing#DNA#
chain.#
As#a#result#of#the#absent#3'#hydroxyl#(-OH)#group#in#a#ddNTP,#no#new#nucleotide#
can#be#added#afterwards
•
DNA#polymerization#stops#once#a#ddNTP#is#added#to#a#growing#stran
Stop#elongation#
○
•
Each#ddNTP#has#a#distinct#fluorescent#label#for#each#base#(A,#G,#C,#T)
Each#ddNTP#is#linked#to#a#fluorescent#molecule
○
Tells#us#about#the#base#found#in#that#given#ddNTP
○
•
Sequencing begins by denaturing template DNA.
The#single-stranded#DNA#is#mixed#with#DNA#polymerase,#primers,#the#4#normal#
dNTPs,#and#small#amounts#of#the#4#fluorescently#tagged#ddNTPs.#
•
In#solution,#DNA#polymerase#synthesizes#strands#of#DNA#using#mostly#the#
normal#dNTPs
•
Once#DNA#polymerase#adds#a#ddNTP#to#the#growing#DNA#strand,#growth#stops.#•
The#result#is#a#solution#with#template#DNA#strands#and#many#shorter#
complementary#strands,#each#one#ending#with#a#fluorescently#tagged#ddNTP
Randomly#cuts#and#has#fragments#of#different#sizes
○
•
Gray: template•
Yellow:#primers•
Red:#normal#dNTPs•
Green/purple/blue:#ddNTP#•
The#new#strands#are#denatured#from#the#templates#and#separated#by#length.#
Form#of#electrophoresis
Rather#than#separating#on#an#agarose#gel,#they're#moved#through#a#thin#
glass#capillary#inside#a#sequencing#machine#that#allows#the#DNA#molecules#
to#flow#through#a#capillary#tube#in#front#of#a#scanner#that#detects#the#
fluorescence#of#each#fragment
○
•
The#shortest#fragments#should#be#one#base#longer#than#the#primer#strand#•
The#color#of#the#fluorescent#tag#at#this#end#of#the#fragments#indicates#the#type#
of#ddNTP#that#was#added#at#the#3'#end#of#the#fragment
•
The#ddNTP#is#ddGTP,#therefore#the#base#on#the#templates#strand#(after#the#primer#
sequence)#is#C
The#remainder#of#the#bases#on#the#template#strand#are#determined#in#a#similar#
manner
•
Ends#up#in#results##as#a#series#of#peaks
Peaks#show#confidence
Higher#=#more#confident#because#=#more#fluorescence#=#higher#
concentration#of#DNA#fragments
§
More#molecules#formed#by#the#random#cuts
More#likely#that#is#the#actual#base#in#the#template□
§
○
•
3. Since DNA sequencing is based on termination of nucleic acid
polymerization, why are normal deoxynucleotides (dNTPs) also included in the
reaction?
To enhance the chain termination ability of the deoxynucleotides
A.
To provide a substrate for DNA polymerase
B.
To produce a range of DNA synthesis products that terminate at
every occurrence of a particular base
Would only get info about one position –the one at the end of the
primer
○
C.
To create DNA synthesis products long enough to allow running a gel
D.
Normal deoxynucleotides are not required in the reaction
E.
The Shotgun Sequencing Process (7 Steps)
Shotgun sequencing involves breaking up the genome into a set of
overlapping fragments that are sequenced, and these sequences are then
put into the correct order.
Manage bits of chromosomes so you can sequence the smaller pieces
and reconstruct the chromosomes on a computer
•
An approach for sequencing the whole genome
•
DNA polymerase can only extend so far
Good at expanding fragments of 1 kb
○
Need to sequence a whole chromosome, can't just use 2 primers
because the genome is too big and the Sanger Sequence primers
are too limited
○
•
Design primers based on pUC sequence
•
The Shotgun Sequencing:
1. Sonication (use of high-frequency sound waves) breaks a genome into
pieces approximately 160 kilobases long
Randomly breaking chromosome in different positions with sound waves
•
The shotgun part
•
2.##Each piece is inserted into a plasmid called a bacterial artificial
chromosome (BAC). A BAC library is created by inserting each BAC into a
different E. coli cell. Colonies of each cell are allowed to grow, creating multiple
copies of each BAC
Can't put 160 kb fragments into a plasmid like pUC
•
BAC has sequences that allow really big pieces of DNA to be replicated
in the DNA of a bacteria
•
3. Each 160-kb DNA segment is broken into 1-kb segments.
4. Each 1-kb segment is cloned into a bacterial plasmid to be sequenced.
These plasmids are then inserted into E. coli cells and replicated, producing
shotgun clones.
5. The fragments from each shotgun clone are then sequenced and analyzed
by computer programs. Regions of overlap between different shotgun clones
are identified.
Allows#you#to#see#how#sequences#fit#together#in#the#chromosome#•
•
6.#Using#regions#of#overlap,#the#computer#puts#the#sequences#in#order,#thus#
reconstructing#the#BACs
Join#the#flanking#left#hand#sequence#with#the#flanking#right#hand#sequence#to#
the#region#of#overlap
•
7. The ends of the reconstructed BACs are similarly analyzed. The goal is to
arrange each 160-kb segment in its correct position along the chromosome,
based on regions of overlap
Learning Catalytics
4.#In#the#shotgun#genome#sequencing#approach,#160#kb#genomic#DNA#fragments#are#
broken#into#1#kb#fragments#(in#“shotgun#clones”)#that#are#sequenced.#
Why#is#this#step#required#(ie—why#not#use#the#160#kb#genomic#DNA#fragments#as#
templates#for#sequencing#reactions)?#
ANSWER:#160#kb#is#too#long#to#sequence#with#Sanger,#need#smaller#fragments#and#
lots#of#breakage#so#you#can#get#pieces#of#overlap
5.#How#are#the#DNA#sequences#of#the#many#different#1#kb#fragments#in#shotgun#clones#
used#to#reconstruct#the#original#160#kb#genomic#DNA#fragments?#
ANSWER: with regions of overlap
Once#a#genome#has#been#sequenced,#how#can#we#determine#which#bases#constitute#
genes?#
Identifying#genes#is#relatively#straightforward#in#prokaryotes#but#is#much#more#
difficult#in#eukaryotes,#who#have#many#noncoding#sequences#in#their#genomes.#
•
Prokaryotes:#use#computer#to#scan#the#DNA#for#long#sequences#not#interrupted#
by#stop#codons
Genes#have#long#stretches#that#start#with#a#start#codon#and#end#in#a#stop
○
A#sequence#with#very#frequent#stop#codons#are#less#likely#to#be#genes
○
•
Computer#programs#are#used#to#scan#a#prokaryotic#genome#sequence#in#both#
directions#in#order#to#identify#open#reading#frames#(ORFs)—long#stretches#of#
sequence#that#lack#a#stop#codon#but#are#flanked#by#a#start#codon#and#a#stop#
codon.#
6#possible#reading#frames#in#a#double#stranded#DNA#molecule
First#reading#frame#incorporates#first#codon,#then#the#following#
codons#are#read
5'#ATGCTAGCGC#-3'□
§
Can#also#ignore#the#first#base#and#start#the#codon#one#over
5'#ATGCTAGCGC#-3'□
§
Can#ignore#first#2#bases
5'#ATGCTAGCGC#- 3'□
§
○
Allows#you#to#identify#open#reading#frames
○
ORFs#are#potential genes#based#on#characteristic#of#being#long#and#not#
interrupted#by#stops
○
Next#step#is#determining#if#the#ORF#is#a#gene
Expected#it#to#be#transcribed
§
○
•
6.#What#evidence#would#you#need#to#conclude#that#an#ORF#is#a#functional#gene?#
ANSWER:#If#you#eliminate#ORF,#that's#a#loss#of#function#experiment#and#expect#a#
mutant#phenotype
In#eukaryotic#organisms,#genes#contain#introns,#and#most#of#the#genome#does#not#
code#for#a#product—thus,#it#is#not#possible#to#scan#for#ORFs
In#eukaryotes,#the#presence#of#introns#complicate#scanning#eukaryotic#genomes#
because#you'll#identify#meaningless#stop#codons
•
The#most#effective#strategy#for#identifying#genes#is#to#use#reverse#transcriptase#
to#produce#a#cDNA#version#of#each#mRNA,#and#sequence#a#portion#of#the#
resulting#molecule#to#produce#an#expressed#sequence#tag,#or#EST
Use#mRNA,#sequence#cDNAs,#look#to#see#where#they#align#in#the#genome
○
Once#you#get#alignment,#you#know#in#this#particular#region#of#the#genome#
that#you#were#able#to#make#a#cDNA
○
•
ESTs#represent#protein-coding#genes•
Genomics)I
Monday,#April#2,#2018
12:01#PM
4.##In#the#shotgun#genome#sequencing#approach,#160#kb#genomic#DNA#fragments#are#
broken#into#1#kb#fragments#(in#“shotgun#clones”)#that#are#sequenced.
Why#is#this#step#required#(ie—why#not#use#the#160#kb#genomic#DNA#fragments#as#
templates#for#sequencing#reactions)?
5. How#are#the#DNA#sequences#of#the#many#different#1#kb#fragments#in#shotgun#
clones#used#to#reconstruct#the#original#160#kb#genomic#DNA#fragments?
Overview
Define#genomics•
Describe#the#use#of#dideoxynucleotide#triphosphates#(ddNTPs)#in#DNA#
sequencing#reactions
•
Explain#the#7#steps#of#shotgun#sequencing•
Explain#how#open#reading#frames#(ORF)#and#expressed#sequence#tags#(EST)#are#
used#to#identify#functional#genes
•
Genomics:#the#scientific#effort#to#sequence,#interpret,#and#compare#whole#genomes
Genome:#the#complete#DNA#sequence#of#an#organism
The#human#genome#sequence#was#published#in#February#2001#as#part#of#
the#Human#Genome#Project
○
•
Genomics:#provides#a#list#of#the#genes#present#in#an#organism•
Functional#genomics:#looks#at#when#those#genes#are#expressed#and#how#their#
products#interact
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
○
•
Which#Genomes#Are#Being#Sequenced,#and#Why?#
The#first#genome#of#an#organism#to#be#sequenced#was#that#of#the#bacterium#
Haemophilus,influenzae,in#1995#it#consists#of#about#1.8#million#base#pairs
Started#with#viruses#because#they#have#a#smaller#genome
○
•
The#first#eukaryotic#genome#to#be#sequenced#was#that#of#the#yeast#
Saccharomyces,cerevisiae,in#1996
Used#as#a#common#model#organism#and#for#commercial#use
Makes#bread#and#beer
§
○
Ferments#in#order#to#rise
○
•
To#date,#complete#genomes#have#been#sequenced#from#lots#of#species.#Most#of#
the#organisms#that#have#been#sequenced#cause#disease#or#have#other#
interesting#biological#properties
•
Dideoxy#DNA#sequencing#was#the#first#sequencing#method#developed#in#1975#by#
Fredrick#Sanger.#
The#method#is#based#on#an#in,vitro,DNA#synthesis#reaction#and#requires#many#of#
the#same#components#used#in#a#PCR#reaction,#including:#
DNA#Template#—something#to#sequence
A.
Deoxyribonucleotides#(dNTPs)#—synthesis#reaction,#need#these#as#
building#blocks#for#the#DNA
B.
DNA#Polymerase
C.
DNA#Primers—provides#3'#ends#for#the#DNA#polymerase#to#incorporate#
bases#on#the#end#of
D.
Dideoxynucleotide#triphosphates#(ddNTPs)#are#also#used#in#addition#to#
the#use#of#dNTPs#in#the#synthesis#reactions
How#this#differs#from#PCR
§
Stops#reaction#from#proceeding#any#further#because#it#lacks#OH#on#
the#C2#and#C3
§
E.
•
Dideoxynucleotide#triphosphates#(ddNTPs)#are#identical#to#dNTPs#except#that#they#
lack#the#3'#hydroxyl#group
dNTP#lacks#a#OH#on#the#C2,#just#has#a#hydrogen
•
ddNTP#lacks#an#OH#on#the#C2#and#C3,#has#2#hydrogens
•
2.#What#happens#when#a#dideoxyribonucleotide#triphosphate#(ddNTP)#is#incorporated#
into#the#3’#end#of#a#growing#DNA#strand#by#DNA#polymerase?#
ddNTPs#permanently#bind#to#the#active#site#of#DNA#polymerase.A.
ddNTPs#cause#DNA#polymerase#to#break#apart#into#small#fragments.#B.
ddNTPs#stop#DNA#polymerization#because#they#lack#a#hydroxyl#(-OH)#group#at#
their#3ʹ#end
Once#you#incorporate#a#ddNTP,#you#no#longer#can#add#any#more#
nucleotides
○
Nothing#for#DNA#polymerase#to#add#another#base#to
○
C.
ddNTPs#have#no#effect#upon#DNA#polymerization.D.
ddNTPs#cause#the#release#of#primers#from#the#template#DNA.#E.
Like#dNTPs,#ddNTPs#are#picked#up#by#DNA#polymerase#and#added#to#a#growing#DNA#
chain.#
As#a#result#of#the#absent#3'#hydroxyl#(-OH)#group#in#a#ddNTP,#no#new#nucleotide#
can#be#added#afterwards
•
DNA#polymerization#stops#once#a#ddNTP#is#added#to#a#growing#stran
Stop#elongation#
○
•
Each#ddNTP#has#a#distinct#fluorescent#label#for#each#base#(A,#G,#C,#T)
Each#ddNTP#is#linked#to#a#fluorescent#molecule
○
Tells#us#about#the#base#found#in#that#given#ddNTP
○
•
Sequencing begins by denaturing template DNA.
The#single-stranded#DNA#is#mixed#with#DNA#polymerase,#primers,#the#4#normal#
dNTPs,#and#small#amounts#of#the#4#fluorescently#tagged#ddNTPs.#
•
In#solution,#DNA#polymerase#synthesizes#strands#of#DNA#using#mostly#the#
normal#dNTPs
•
Once#DNA#polymerase#adds#a#ddNTP#to#the#growing#DNA#strand,#growth#stops.#•
The#result#is#a#solution#with#template#DNA#strands#and#many#shorter#
complementary#strands,#each#one#ending#with#a#fluorescently#tagged#ddNTP
Randomly#cuts#and#has#fragments#of#different#sizes
○
•
Gray: template•
Yellow:#primers•
Red:#normal#dNTPs•
Green/purple/blue:#ddNTP#•
The#new#strands#are#denatured#from#the#templates#and#separated#by#length.#
Form#of#electrophoresis
Rather#than#separating#on#an#agarose#gel,#they're#moved#through#a#thin#
glass#capillary#inside#a#sequencing#machine#that#allows#the#DNA#molecules#
to#flow#through#a#capillary#tube#in#front#of#a#scanner#that#detects#the#
fluorescence#of#each#fragment
○
•
The#shortest#fragments#should#be#one#base#longer#than#the#primer#strand#•
The#color#of#the#fluorescent#tag#at#this#end#of#the#fragments#indicates#the#type#
of#ddNTP#that#was#added#at#the#3'#end#of#the#fragment
•
The#ddNTP#is#ddGTP,#therefore#the#base#on#the#templates#strand#(after#the#primer#
sequence)#is#C
The#remainder#of#the#bases#on#the#template#strand#are#determined#in#a#similar#
manner
•
Ends#up#in#results##as#a#series#of#peaks
Peaks#show#confidence
Higher#=#more#confident#because#=#more#fluorescence#=#higher#
concentration#of#DNA#fragments
§
More#molecules#formed#by#the#random#cuts
More#likely#that#is#the#actual#base#in#the#template□
§
○
•
3. Since DNA sequencing is based on termination of nucleic acid
polymerization, why are normal deoxynucleotides (dNTPs) also included in the
reaction?
To enhance the chain termination ability of the deoxynucleotides
A.
To provide a substrate for DNA polymerase
B.
To produce a range of DNA synthesis products that terminate at
every occurrence of a particular base
Would only get info about one position –the one at the end of the
primer
○
C.
To create DNA synthesis products long enough to allow running a gel
D.
Normal deoxynucleotides are not required in the reaction
E.
The Shotgun Sequencing Process (7 Steps)
Shotgun sequencing involves breaking up the genome into a set of
overlapping fragments that are sequenced, and these sequences are then
put into the correct order.
Manage bits of chromosomes so you can sequence the smaller pieces
and reconstruct the chromosomes on a computer
•
An approach for sequencing the whole genome
•
DNA polymerase can only extend so far
Good at expanding fragments of 1 kb
○
Need to sequence a whole chromosome, can't just use 2 primers
because the genome is too big and the Sanger Sequence primers
are too limited
○
•
Design primers based on pUC sequence
•
The Shotgun Sequencing:
1. Sonication (use of high-frequency sound waves) breaks a genome into
pieces approximately 160 kilobases long
Randomly breaking chromosome in different positions with sound waves
•
The shotgun part
•
2.##Each piece is inserted into a plasmid called a bacterial artificial
chromosome (BAC). A BAC library is created by inserting each BAC into a
different E. coli cell. Colonies of each cell are allowed to grow, creating multiple
copies of each BAC
Can't put 160 kb fragments into a plasmid like pUC
•
BAC has sequences that allow really big pieces of DNA to be replicated
in the DNA of a bacteria
•
3. Each 160-kb DNA segment is broken into 1-kb segments.
4. Each 1-kb segment is cloned into a bacterial plasmid to be sequenced.
These plasmids are then inserted into E. coli cells and replicated, producing
shotgun clones.
5. The fragments from each shotgun clone are then sequenced and analyzed
by computer programs. Regions of overlap between different shotgun clones
are identified.
Allows#you#to#see#how#sequences#fit#together#in#the#chromosome#•
•
6.#Using#regions#of#overlap,#the#computer#puts#the#sequences#in#order,#thus#
reconstructing#the#BACs
Join#the#flanking#left#hand#sequence#with#the#flanking#right#hand#sequence#to#
the#region#of#overlap
•
7. The ends of the reconstructed BACs are similarly analyzed. The goal is to
arrange each 160-kb segment in its correct position along the chromosome,
based on regions of overlap
Learning Catalytics
4.#In#the#shotgun#genome#sequencing#approach,#160#kb#genomic#DNA#fragments#are#
broken#into#1#kb#fragments#(in#“shotgun#clones”)#that#are#sequenced.#
Why#is#this#step#required#(ie—why#not#use#the#160#kb#genomic#DNA#fragments#as#
templates#for#sequencing#reactions)?#
ANSWER:#160#kb#is#too#long#to#sequence#with#Sanger,#need#smaller#fragments#and#
lots#of#breakage#so#you#can#get#pieces#of#overlap
5.#How#are#the#DNA#sequences#of#the#many#different#1#kb#fragments#in#shotgun#clones#
used#to#reconstruct#the#original#160#kb#genomic#DNA#fragments?#
ANSWER: with regions of overlap
Once#a#genome#has#been#sequenced,#how#can#we#determine#which#bases#constitute#
genes?#
Identifying#genes#is#relatively#straightforward#in#prokaryotes#but#is#much#more#
difficult#in#eukaryotes,#who#have#many#noncoding#sequences#in#their#genomes.#
•
Prokaryotes:#use#computer#to#scan#the#DNA#for#long#sequences#not#interrupted#
by#stop#codons
Genes#have#long#stretches#that#start#with#a#start#codon#and#end#in#a#stop
○
A#sequence#with#very#frequent#stop#codons#are#less#likely#to#be#genes
○
•
Computer#programs#are#used#to#scan#a#prokaryotic#genome#sequence#in#both#
directions#in#order#to#identify#open#reading#frames#(ORFs)—long#stretches#of#
sequence#that#lack#a#stop#codon#but#are#flanked#by#a#start#codon#and#a#stop#
codon.#
6#possible#reading#frames#in#a#double#stranded#DNA#molecule
First#reading#frame#incorporates#first#codon,#then#the#following#
codons#are#read
5'#ATGCTAGCGC#-3'□
§
Can#also#ignore#the#first#base#and#start#the#codon#one#over
5'#ATGCTAGCGC#-3'□
§
Can#ignore#first#2#bases
5'#ATGCTAGCGC#- 3'□
§
○
Allows#you#to#identify#open#reading#frames
○
ORFs#are#potential genes#based#on#characteristic#of#being#long#and#not#
interrupted#by#stops
○
Next#step#is#determining#if#the#ORF#is#a#gene
Expected#it#to#be#transcribed
§
○
•
6.#What#evidence#would#you#need#to#conclude#that#an#ORF#is#a#functional#gene?#
ANSWER:#If#you#eliminate#ORF,#that's#a#loss#of#function#experiment#and#expect#a#
mutant#phenotype
In#eukaryotic#organisms,#genes#contain#introns,#and#most#of#the#genome#does#not#
code#for#a#product—thus,#it#is#not#possible#to#scan#for#ORFs
In#eukaryotes,#the#presence#of#introns#complicate#scanning#eukaryotic#genomes#
because#you'll#identify#meaningless#stop#codons
•
The#most#effective#strategy#for#identifying#genes#is#to#use#reverse#transcriptase#
to#produce#a#cDNA#version#of#each#mRNA,#and#sequence#a#portion#of#the#
resulting#molecule#to#produce#an#expressed#sequence#tag,#or#EST
Use#mRNA,#sequence#cDNAs,#look#to#see#where#they#align#in#the#genome
○
Once#you#get#alignment,#you#know#in#this#particular#region#of#the#genome#
that#you#were#able#to#make#a#cDNA
○
•
ESTs#represent#protein-coding#genes•
Genomics)I
Monday,#April#2,#2018 12:01#PM
4.##In#the#shotgun#genome#sequencing#approach,#160#kb#genomic#DNA#fragments#are#
broken#into#1#kb#fragments#(in#“shotgun#clones”)#that#are#sequenced.
Why#is#this#step#required#(ie—why#not#use#the#160#kb#genomic#DNA#fragments#as#
templates#for#sequencing#reactions)?
5. How#are#the#DNA#sequences#of#the#many#different#1#kb#fragments#in#shotgun#
clones#used#to#reconstruct#the#original#160#kb#genomic#DNA#fragments?
Overview
Define#genomics•
Describe#the#use#of#dideoxynucleotide#triphosphates#(ddNTPs)#in#DNA#
sequencing#reactions
•
Explain#the#7#steps#of#shotgun#sequencing•
Explain#how#open#reading#frames#(ORF)#and#expressed#sequence#tags#(EST)#are#
used#to#identify#functional#genes
•
Genomics:#the#scientific#effort#to#sequence,#interpret,#and#compare#whole#genomes
Genome:#the#complete#DNA#sequence#of#an#organism
The#human#genome#sequence#was#published#in#February#2001#as#part#of#
the#Human#Genome#Project
○
•
Genomics:#provides#a#list#of#the#genes#present#in#an#organism•
Functional#genomics:#looks#at#when#those#genes#are#expressed#and#how#their#
products#interact
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
○
•
Which#Genomes#Are#Being#Sequenced,#and#Why?#
The#first#genome#of#an#organism#to#be#sequenced#was#that#of#the#bacterium#
Haemophilus,influenzae,in#1995#it#consists#of#about#1.8#million#base#pairs
Started#with#viruses#because#they#have#a#smaller#genome
○
•
The#first#eukaryotic#genome#to#be#sequenced#was#that#of#the#yeast#
Saccharomyces,cerevisiae,in#1996
Used#as#a#common#model#organism#and#for#commercial#use
Makes#bread#and#beer
§
○
Ferments#in#order#to#rise
○
•
To#date,#complete#genomes#have#been#sequenced#from#lots#of#species.#Most#of#
the#organisms#that#have#been#sequenced#cause#disease#or#have#other#
interesting#biological#properties
•
Dideoxy#DNA#sequencing#was#the#first#sequencing#method#developed#in#1975#by#
Fredrick#Sanger.#
The#method#is#based#on#an#in,vitro,DNA#synthesis#reaction#and#requires#many#of#
the#same#components#used#in#a#PCR#reaction,#including:#
DNA#Template#—something#to#sequenceA.
Deoxyribonucleotides#(dNTPs)#—synthesis#reaction,#need#these#as#
building#blocks#for#the#DNA
B.
DNA#PolymeraseC.
DNA#Primers—provides#3'#ends#for#the#DNA#polymerase#to#incorporate#
bases#on#the#end#of
D.
Dideoxynucleotide#triphosphates#(ddNTPs)#are#also#used#in#addition#to#
the#use#of#dNTPs#in#the#synthesis#reactions
How#this#differs#from#PCR
§
Stops#reaction#from#proceeding#any#further#because#it#lacks#OH#on#
the#C2#and#C3
§
E.
•
Dideoxynucleotide#triphosphates#(ddNTPs)#are#identical#to#dNTPs#except#that#they#
lack#the#3'#hydroxyl#group
dNTP#lacks#a#OH#on#the#C2,#just#has#a#hydrogen•
ddNTP#lacks#an#OH#on#the#C2#and#C3,#has#2#hydrogens•
2.#What#happens#when#a#dideoxyribonucleotide#triphosphate#(ddNTP)#is#incorporated#
into#the#3’#end#of#a#growing#DNA#strand#by#DNA#polymerase?#
ddNTPs#permanently#bind#to#the#active#site#of#DNA#polymerase.
A.
ddNTPs#cause#DNA#polymerase#to#break#apart#into#small#fragments.#
B.
ddNTPs#stop#DNA#polymerization#because#they#lack#a#hydroxyl#(-OH)#group#at#
their#3ʹ#end
Once#you#incorporate#a#ddNTP,#you#no#longer#can#add#any#more#
nucleotides
○
Nothing#for#DNA#polymerase#to#add#another#base#to
○
C.
ddNTPs#have#no#effect#upon#DNA#polymerization.
D.
ddNTPs#cause#the#release#of#primers#from#the#template#DNA.#
E.
Like#dNTPs,#ddNTPs#are#picked#up#by#DNA#polymerase#and#added#to#a#growing#DNA#
chain.#
As#a#result#of#the#absent#3'#hydroxyl#(-OH)#group#in#a#ddNTP,#no#new#nucleotide#
can#be#added#afterwards
•
DNA#polymerization#stops#once#a#ddNTP#is#added#to#a#growing#stran
Stop#elongation#
○
•
Each#ddNTP#has#a#distinct#fluorescent#label#for#each#base#(A,#G,#C,#T)
Each#ddNTP#is#linked#to#a#fluorescent#molecule
○
Tells#us#about#the#base#found#in#that#given#ddNTP
○
•
Sequencing begins by denaturing template DNA.
The#single-stranded#DNA#is#mixed#with#DNA#polymerase,#primers,#the#4#normal#
dNTPs,#and#small#amounts#of#the#4#fluorescently#tagged#ddNTPs.#
•
In#solution,#DNA#polymerase#synthesizes#strands#of#DNA#using#mostly#the#
normal#dNTPs
•
Once#DNA#polymerase#adds#a#ddNTP#to#the#growing#DNA#strand,#growth#stops.#•
The#result#is#a#solution#with#template#DNA#strands#and#many#shorter#
complementary#strands,#each#one#ending#with#a#fluorescently#tagged#ddNTP
Randomly#cuts#and#has#fragments#of#different#sizes
○
•
Gray: template•
Yellow:#primers•
Red:#normal#dNTPs•
Green/purple/blue:#ddNTP#•
The#new#strands#are#denatured#from#the#templates#and#separated#by#length.#
Form#of#electrophoresis
Rather#than#separating#on#an#agarose#gel,#they're#moved#through#a#thin#
glass#capillary#inside#a#sequencing#machine#that#allows#the#DNA#molecules#
to#flow#through#a#capillary#tube#in#front#of#a#scanner#that#detects#the#
fluorescence#of#each#fragment
○
•
The#shortest#fragments#should#be#one#base#longer#than#the#primer#strand#•
The#color#of#the#fluorescent#tag#at#this#end#of#the#fragments#indicates#the#type#
of#ddNTP#that#was#added#at#the#3'#end#of#the#fragment
•
The#ddNTP#is#ddGTP,#therefore#the#base#on#the#templates#strand#(after#the#primer#
sequence)#is#C
The#remainder#of#the#bases#on#the#template#strand#are#determined#in#a#similar#
manner
•
Ends#up#in#results##as#a#series#of#peaks
Peaks#show#confidence
Higher#=#more#confident#because#=#more#fluorescence#=#higher#
concentration#of#DNA#fragments
§
More#molecules#formed#by#the#random#cuts
More#likely#that#is#the#actual#base#in#the#template□
§
○
•
3. Since DNA sequencing is based on termination of nucleic acid
polymerization, why are normal deoxynucleotides (dNTPs) also included in the
reaction?
To enhance the chain termination ability of the deoxynucleotides
A.
To provide a substrate for DNA polymerase
B.
To produce a range of DNA synthesis products that terminate at
every occurrence of a particular base
Would only get info about one position –the one at the end of the
primer
○
C.
To create DNA synthesis products long enough to allow running a gel
D.
Normal deoxynucleotides are not required in the reaction
E.
The Shotgun Sequencing Process (7 Steps)
Shotgun sequencing involves breaking up the genome into a set of
overlapping fragments that are sequenced, and these sequences are then
put into the correct order.
Manage bits of chromosomes so you can sequence the smaller pieces
and reconstruct the chromosomes on a computer
•
An approach for sequencing the whole genome
•
DNA polymerase can only extend so far
Good at expanding fragments of 1 kb
○
Need to sequence a whole chromosome, can't just use 2 primers
because the genome is too big and the Sanger Sequence primers
are too limited
○
•
Design primers based on pUC sequence
•
The Shotgun Sequencing:
1. Sonication (use of high-frequency sound waves) breaks a genome into
pieces approximately 160 kilobases long
Randomly breaking chromosome in different positions with sound waves
•
The shotgun part
•
2.##Each piece is inserted into a plasmid called a bacterial artificial
chromosome (BAC). A BAC library is created by inserting each BAC into a
different E. coli cell. Colonies of each cell are allowed to grow, creating multiple
copies of each BAC
Can't put 160 kb fragments into a plasmid like pUC
•
BAC has sequences that allow really big pieces of DNA to be replicated
in the DNA of a bacteria
•
3. Each 160-kb DNA segment is broken into 1-kb segments.
4. Each 1-kb segment is cloned into a bacterial plasmid to be sequenced.
These plasmids are then inserted into E. coli cells and replicated, producing
shotgun clones.
5. The fragments from each shotgun clone are then sequenced and analyzed
by computer programs. Regions of overlap between different shotgun clones
are identified.
Allows#you#to#see#how#sequences#fit#together#in#the#chromosome#•
•
6.#Using#regions#of#overlap,#the#computer#puts#the#sequences#in#order,#thus#
reconstructing#the#BACs
Join#the#flanking#left#hand#sequence#with#the#flanking#right#hand#sequence#to#
the#region#of#overlap
•
7. The ends of the reconstructed BACs are similarly analyzed. The goal is to
arrange each 160-kb segment in its correct position along the chromosome,
based on regions of overlap
Learning Catalytics
4.#In#the#shotgun#genome#sequencing#approach,#160#kb#genomic#DNA#fragments#are#
broken#into#1#kb#fragments#(in#“shotgun#clones”)#that#are#sequenced.#
Why#is#this#step#required#(ie—why#not#use#the#160#kb#genomic#DNA#fragments#as#
templates#for#sequencing#reactions)?#
ANSWER:#160#kb#is#too#long#to#sequence#with#Sanger,#need#smaller#fragments#and#
lots#of#breakage#so#you#can#get#pieces#of#overlap
5.#How#are#the#DNA#sequences#of#the#many#different#1#kb#fragments#in#shotgun#clones#
used#to#reconstruct#the#original#160#kb#genomic#DNA#fragments?#
ANSWER: with regions of overlap
Once#a#genome#has#been#sequenced,#how#can#we#determine#which#bases#constitute#
genes?#
Identifying#genes#is#relatively#straightforward#in#prokaryotes#but#is#much#more#
difficult#in#eukaryotes,#who#have#many#noncoding#sequences#in#their#genomes.#
•
Prokaryotes:#use#computer#to#scan#the#DNA#for#long#sequences#not#interrupted#
by#stop#codons
Genes#have#long#stretches#that#start#with#a#start#codon#and#end#in#a#stop
○
A#sequence#with#very#frequent#stop#codons#are#less#likely#to#be#genes
○
•
Computer#programs#are#used#to#scan#a#prokaryotic#genome#sequence#in#both#
directions#in#order#to#identify#open#reading#frames#(ORFs)—long#stretches#of#
sequence#that#lack#a#stop#codon#but#are#flanked#by#a#start#codon#and#a#stop#
codon.#
6#possible#reading#frames#in#a#double#stranded#DNA#molecule
First#reading#frame#incorporates#first#codon,#then#the#following#
codons#are#read
5'#ATGCTAGCGC#-3'□
§
Can#also#ignore#the#first#base#and#start#the#codon#one#over
5'#ATGCTAGCGC#-3'□
§
Can#ignore#first#2#bases
5'#ATGCTAGCGC#- 3'□
§
○
Allows#you#to#identify#open#reading#frames
○
ORFs#are#potential genes#based#on#characteristic#of#being#long#and#not#
interrupted#by#stops
○
Next#step#is#determining#if#the#ORF#is#a#gene
Expected#it#to#be#transcribed
§
○
•
6.#What#evidence#would#you#need#to#conclude#that#an#ORF#is#a#functional#gene?#
ANSWER:#If#you#eliminate#ORF,#that's#a#loss#of#function#experiment#and#expect#a#
mutant#phenotype
In#eukaryotic#organisms,#genes#contain#introns,#and#most#of#the#genome#does#not#
code#for#a#product—thus,#it#is#not#possible#to#scan#for#ORFs
In#eukaryotes,#the#presence#of#introns#complicate#scanning#eukaryotic#genomes#
because#you'll#identify#meaningless#stop#codons
•
The#most#effective#strategy#for#identifying#genes#is#to#use#reverse#transcriptase#
to#produce#a#cDNA#version#of#each#mRNA,#and#sequence#a#portion#of#the#
resulting#molecule#to#produce#an#expressed#sequence#tag,#or#EST
Use#mRNA,#sequence#cDNAs,#look#to#see#where#they#align#in#the#genome
○
Once#you#get#alignment,#you#know#in#this#particular#region#of#the#genome#
that#you#were#able#to#make#a#cDNA
○
•
ESTs#represent#protein-coding#genes•
Genomics)I
Monday,#April#2,#2018 12:01#PM
Document Summary
12:01 pm: in the shotgun genome sequencing approach, 160 kb genomic dna fragments are broken into 1 kb fragments (in shotgun clones ) that are sequenced. Describe the use of dideoxynucleotide triphosphates (ddntps) in dna sequencing reactions. Explain how open reading frames (orf) and expressed sequence tags (est) are used to identify functional genes. Genomics: the scientific effort to sequence, interpret, and compare whole genomes. Genome: the complete dna sequence of an organism. The human genome sequence was published in february 2001 as part of the human genome project. Genomics: provides a list of the genes present in an organism. Functional genomics: looks at when those genes are expressed and how their products interact. When and where are genes being turned on a making products. Proteomics: study of proteins and how they work in a large scale. The first genome of an organism to be sequenced was that of the bacterium.
