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Genomics
Chapter 22
What is a Genome?
• It is the total DNA content in a typical cell
of an organism
• In multicellular organisms, most cells will
have exact same genome inside
• Exceptions:
– Humans red blood cells which lack nuclei
– Sperm and ova which have haploid genomes
– Cancer-transformed cells which may have
amplified or deleted genes
Genomics
Studying the genome:
• Karyotyping: (largest)
– Examining chromosomes and banding
• Linkage mapping:
– Genetic distances (cM), position of markers
• Physical mapping:
– Physical distances (Mb), position of genes
• Sequencing: (smallest)
– Actual order of bases
Levels of Mapping
Sequencing
• Sequencing can only be done with about
800 bases at a time
• After this it starts to fall apart, no longer
read with any accuracy
• Sequencing an entire genome is done by
first shredding DNA apart
• Then trying to piece it back together 800
bases at a time
• Encyclopedia analogy
Sanger Method
1977
• Fredrick Sanger
• Dideoxy sequencing
• Dideoxys are nucleotides that contain no
free oxygen at all
– These nucleotides cannot form chains
– Polymerase stops copying DNA’s sequence
when it adds a dideoxy base
– Make each nucleotide with dideoxy sugar
Sanger Method
• Dideoxy nucleotides:
OH
OH
OH
ribose
HO-CH2
OH
HO-CH2
OH
OH
HO-CH2
deoxyribose
dideoxyribose
Sanger Method
• In four tubes add:
– DNA of unknown sequence
– Everything necessary for DNA replication
– One of each dideoxy nucleotide
• Each tube has a different dideoxy nucleotide
in it (A, C, G or T)
• DNA polymerase will stop working once it
adds a dideoxy base
• Therefore, get different lengths of copied DNA
Sanger Method
• Run each separate tube in it’s own lane on
a gel
– “A” lane, the “T” lane, etc
• Important – some regular nucleotide is also
added so that sequence can continue past
some of the time
• Fragments of different lengths
• Read the four lanes to determine sequence
of complete DNA fragment
Sanger Method
Modern Sequencing
• Added florescent dyes:
– A is red
– C is blue
– T is green
– G is yellow
• Now can run all four tubes in same lane
• Automate entire process:
– Invented an automated sequencer and a
computer program to read the gels
Modern Sequencing
Practice Interpreting Sanger
• Determine the sequence from this gel:
dd-A
dd-C
dd-T
dd-G
TGCACTGAATCAGTGCT
ACGTGACTTAGTCACGA
Direct Read
Actual Sequence
Building sequences
• Based on overlapping fragments
Building sequences
• Based on overlapping fragments
Why 8X Coverage?
When sequencing the complete genome of
any organism (humans included) they
always use DNA from 6 to 8 different
individuals – WHY?
• Ensure fragments will overlap often
• Ensure each base is covered with at least
two good clean “reads”
• Identify common polymorphisms
Important Pieces:
Need to know:
• Dideoxy nucleotides
• STSs
• BACs
– NIH and International Consortium’s (Public)
Genome Project
• ESTs
– Celera’s Genome Project
STS = Sequence Tagged Sites
Short sequences that are completely unique
in the genome:
• Already mapped to exact physical position
in the genome
• 20 to 30 bases long
• Genomewide sequencing uses STS to
identify where a given sequence lies within
genome
• Sort of like Road Signs
BAC = Bacterial Artificial
Chromosome
Cloning vector to hold fragments of DNA
• Entire genome divided into BACs
– Each BAC can hold ~100,000 bases
• Know which chromosomal regions are in
which BACs
• BACs then sequenced:
– 800 bases at a time
• Sequences overlapped and built up
EST = Expressed Sequence Tag
A small piece of DNA that is known to be
expressed in certain cell type
• Therefore ESTs represent only DNA that
encodes proteins
• EST libraries have been developed and
can be checked against:
– For different organisms or cell types
• Now can focus on only sequencing protein
coding regions of genome
Human Genome Project
• Public Consortium:
– Used BACs
– Posted their sequencing results every night
on GenBank
• Celera Genomics:
– Skipped the BACs and shotgunned the entire
genome into fragments
– Only sequenced EST positive fragments
– Used STSs to align sequences at the end
– Updated their analysis from GenBank every
morning
Human Genome Currently
• All protein coding regions are completely
sequenced and aligned
• Many non-coding regions are sequenced
but unaligned
• Many repeat regions still unsequenced
• Annotation is ongoing:
– Determining where genes are
– Determining gene function
– Determining gene involvement in diseases
Positional Cloning
Gene mapping:
• Begin with pedigrees of affecteds
• Step One – Linkage
– Identify regions of genome where gene lies
• Step Two – Fine Mapping
– Use more markers to determine exactly where
gene is on chromosome
• Step Three – Gene Identification
– Pinpoint exact gene causing disease
Positional Cloning
• Time consuming
• Only works for Mendelian disorders
• However it does work:
– Cystic Fibrosis
– Huntington’s Disease
– Early onset Alzheimer's
• Sort of the opposite of Human Genome
project
– “Sequence now – Interpret later” – HGP
Positional Cloning
• Still happening today
• Book says “…a graduate student can find
a gene in weeks”
• Not true – even with entire genome
sequenced, even with Mendelian disorders
• Still have to analyze the sequences and
identify which genes are involved in which
disorders
• Not to mention complex disorders…
What is more important?
• Sequencing the entire human genome or
positional cloning all the genes?
• Should the non-coding regions be
sequenced? Why or why not?
• What about all the annotation?
– Genome comparisons (to model organisms)
– Identifying gene positions
– Identifying gene function
• Focus on Mendelian or Complex disorders?
Genome Comparisons
• The human genome is 3 x 109 base pairs
(3000 Mb)
• Prokaryotic genomes between 1 - 6 x 106
• Model organisms
– S. cerevisiae (yeast)
– C. elegans (roundworm)
– D. melanogaster (fruit fly)
– M. musculus (mouse)
12 Mb
100 Mb
170 Mb
3000 Mb
• Some plant genomes are much larger
(e.g., onion is 15000 Mb)
Genome Comparisons
•
•
•
•
Larger genome doesn’t mean more genes
Prokaryotes have about 1 to 6 1000 genes
Yeast has about 6,000 genes
But most multicellular organisms (animals
and plants) have around 30,000
• Humans included
• Rest of space in genome?
– Repeats (especially in plants)
– More/larger introns in genes
Synteny
Comparing genomes
shows regions of
synteny
Allows faster
identification
of homologous genes
Human chromosomes
with mouse pieces
labeled
Bioinformatics
• Compare sequences to model organisms:
– Determine gene function (homology)
• Search for gene positions:
– Known genes are labeled on Human Genome
Browser (www.genome.ucsc.edu)
– Gene-like sequences are searched for to try
to identify position of unknown genes
• Gene expression profiles:
– Determine which genes are expressed when
• Gene pathways/networks
Example – post HGP:
Searching for the genes involved in Autism
Disorder:
1. Linkage Analysis
– Use 345 pedigrees to find linkage
2. Fine Mapping
– Narrow down region of linkage
3. Bioinformatics
– Determine what genes exist in linked region
4. Follow up on a candidate gene
Outcomes of HGP
Summary
Know:
• How Sanger (dideoxy) sequencing works
• Modern changes to sequencing
• BACs, ESTs, STSs
• Positional Cloning basics
• Genome comparison generalizations
• What bioinformatics is/can determine
Ethics Discussion
• Get into groups of 3 or 4
• Discuss the following ethical decisions
regarding genome sequencing
• Make notes of your discussions
Genome Ethics
1. Would you want to know all the diseases
that you are predisposed for?
Why or why not?
What if the disease has a cure?
What if there is only a painful treatment that
works in 30% of people, but no cure?
Should parents be allowed to determine
their minor children’s predispositions?
Genome Ethics
2. Lets say that personalized medicine is a
real possibility, but it still costs 10,000
dollars to sequence your genome.
Who should pay the cost?
You, your insurance, the government?
How is that information kept private?
What sort of diseases would be worth
spending the money for? Which ones
wouldn’t make sense?
Genome Ethics
3. Lets say the genome can be sequenced
for $1,000. What if people could get their
analysis through the mail, without every
talking to a doctor.
What are the pros and cons of this situation?
What are some potential fall outs of receiving
your genome analysis this way?
How should the law control this procedure?
Genome Ethics
4. Think about behavioral disorders such as
depression, anxiety or schizophrenia?
Where is the line between treatment and
medical enhancement?
Who should enforce this line?
Will things change once we learn all the
genes involved in specific disorders?
What about homosexuality?
Next Class:
• Homework – Chapter 22 Problems;
– Review: 1, 3, 4
– Applied: 1, 5, 6, 9, 11
– Also – write out at least 5 questions about
material to review on Wednesday
• Review All Chapters and Notes and Exams
Next Class:
Review Chapters 1, 3-18, 20-22
• Go through your review questions
• Final Exam:
Twice as long as regular tests (200 pts)
Cumulative
Monday – December 12th – 8 pm
Test
dd-A
dd-C
dd-T
dd-G