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The project of mapping Human
Genome
•Why they want to make
a map of the human
genome ?????
The project of mapping Human
Genome
• The objective of sequencing human genome:
1. To understand how genes work together to direct
the growth, development and maintenance of an
entire organism.
2. By knowing the whole genome sequence it will
help to study the parts of the genome outside the
genes. This includes the long sequences of nonsense
(junk) DNA that has no clear functions.
3. To learn about other important parts of the
genome, such as the regulatory regions that help
control when genes are turned on and off.
4. To draw accurate map for the chromosomal
locations of genes responsible for genetic diseases.
Already, about 1400 genes are identified for human
genetic diseases as a result of human genome
mapping.
5. By comparing human genome map with other
species maps, it will be possible to understand the
process of evolution.
The plan
The project of human genome sequencing
began on 1990 and completed on 2003. The
whole genome cannot be sequenced all at once
because available methods of DNA sequencing
can only work with short stretches of DNA at a
time. Instead, the genome was broken into
smaller pieces; approximately 150,000 base
pairs in length. These pieces were cloned into
plasmid vector before they were amplifies in
bacterial culture.
• Using restriction enzymes, the pieces of
human DNA were cut into small pieces and
each gene was identified by specific probe
before it was sequenced. Then the genes were
reassembled in the proper order to obtain the
sequence of the whole genome.
Data obtained from mapping human genome
1. The human genome sequence is almost exactly the
same (99.9%) in all people.
2. There are approximately 23,000 genes in human
beings being mapped, the same range as in mice
and roundworms. Before this mapping process, the
human genome was estimated to contain about
80-140 thousands genes, based on comparison with
the size of bacteria in which the actual gene
mapping already obtained.
3. The human genome contains 3.2 billion nucleotide base
pairs .The average gene consists of 3,000 base pairs, but
sizes vary greatly, with the largest known human gene has
2.4 million base pairs which is responsible for expressing
the dystrophin protein.
4. Functions are still unknown for more than 50% of
discovered genes.
5. Genes appear to be concentrated in random areas
along the genome, with extended regions of
non-coding DNA in between.
6. Particular gene sequences have been found to be
associated with various diseases, including breast
cancer, muscle disease, deafness, and blindness.
7. Pieces of up to 30,000 C and G bases repeating over
and over often occur adjacent to gene-rich areas,
forming a barrier between the genes and the junk
DNA. These C-G rich segments are believed to help in
the regulation of gene activity.
8.Chromosome 1 (the largest human chromosome)
has the most number of genes (3,168), and Y
chromosome has the fewest number of genes (344).
Comparison of human genome with other organisms
1 . Unlike the random distribution of gene‐rich areas
in human's, many other organisms'
genomes are more uniform, with genes evenly
spaced throughout.
2. Humans have on average three times as many
kinds of proteins as the fly or worm because of
mRNA transcript alternative splicing and chemical
modifications to the proteins. This process can yield
different protein products from the same gene.
3. Humans share similar protein families with
worms, flies, and plants, but the number of gene
family members are more expanded in humans,
especially in proteins involved in development and
immunity.
4. The human genome has a much greater portion
(50%) of repeat sequences than the mustard weed
plant (11%), the worm (7%), and the fly (3%).
5. Over 40% of predicted human proteins share
similarity with fruit‐fly or worm proteins
6. As a conclusion from this mapping sequence
of human genome, it is believed that the quality
of protein types produced by the genome is
more important in providing the overall human
phenotype than the number of genes that
express these proteins.
DNA sequencing
Why sequence DNA?
• All genes available for an organism to use -- a
very important tool for biologists
• Not just sequence of genes, but also positioning
of genes and sequences of regulatory regions
• New recombinant DNA constructs must be
sequenced to verify construction or positions of
mutations
Sequencing Methods
•
•
•
•
•
Maxam/Gilbert chemical sequencing
Sanger chain termination sequencing
Pyrosequencing
Bisulfite Sequencing
Array sequencing
Maxam-Gilbert Sequencing
A.
Maxam-Gilbert chemical cleavage method: DNA is labelled and
then chemically cleaved in a sequence-dependent manner. This
method is not easily scaled and is rather tedious
DMS
FA
G
H
G
G
H+S
C
A
G
T
T
G
G
G
C
A
G
C
C
C
C
T
C
A
A
C
T
Maxam-Gilbert sequencing is performed by chain
breakage at specific nucleotides.
Maxam-Gilbert Sequencing
G
Longer fragments
A
Shortest fragments
G
G+A T+C
C
3′
A
A
G
C
A
A
C
G
T
G
C
A
G
5′
Sequencing gels are read from bottom to top (5′ to 3′).
Chain Termination (Sanger) Sequencing
Sanger dideoxy (primer extension/chain-termination)
method: most popular protocol for sequencing, very
adaptable, scalable to large sequencing projects
The 3′-OH group necessary for formation of the
phosphodiester bond is missing in ddNTPs.
Chain terminates
at ddG
Chain Termination (Sanger) Sequencing
• A sequencing reaction mix includes labeled primer
and template.
Primer
-3′ OH
TCGACGGGC…
5′OPTemplate
Template area to be sequenced
• Dideoxynucleotides are added separately to each of
the four tubes.
Chain Termination (Sanger) Sequencing
A
C
ddATP +
four dNTPs
ddA
dAdGdCdTdGdCdCdCdG
ddCTP +
four dNTPs
dAdGddC
dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdCddC
ddGTP +
G four dNTPs
T
ddTTP +
four dNTPs
dAddG
dAdGdCdTddG
dAdGdCdTdGdCdCdCddG
dAdGdCddT
dAdGdCdTdGdCdCdCdG
Chain Termination (Sanger) Sequencing
• With addition of enzyme (DNA polymerase),
the primer is extended until a ddNTP is
encountered.
• The chain will end with the incorporation of
the ddNTP.
• With the proper dNTP:ddNTP ratio, the chain
will terminate throughout the length of the
template.
• All terminated chains will end in the ddNTP
added to that reaction.
Chain Termination (Sanger) Sequencing
• The collection of fragments is a
sequencing ladder.
• The resulting terminated chains are
resolved by electrophoresis.
• Fragments from each of the four tubes
are placed in four separate gel lanes.
Chain Termination (Sanger) Sequencing
Longer fragments
ddG
Shorter fragments
ddG
G
A
T
C
3′
G
G
T
A
A
A
T
C
A
T
G
5′
Sequencing gels are read from bottom to top (5′ to 3′).
Chain Termination (Sanger) Sequencing
• A modified DNA
replication reaction.
• Growing chains are
terminated by
dideoxynucleotides.
for dideoxy sequencing you need:
1) Single stranded DNA template
2) A primer for DNA synthesis
3) DNA polymerase
4) Deoxynucleoside triphosphates and
dideoxynucleotide triphosphates
Primers for DNA sequencing
• Oligonucleotide primers can be synthesized by
phosphoramidite chemistry--usually designed
manually and then purchased
• Sequence of the oligo must be complimentary to
DNA flanking sequenced region
• Oligos are usually 15-30 nucleotides in length
DNA templates for sequencing:
• Single stranded DNA isolated from recombinant
M13 bacteriophage containing DNA of interest
• Double-stranded DNA that has been denatured
• Non-denatured double stranded DNA (cycle
sequencing)
Reagents for sequencing:
DNA polymerases
• Should be highly processive, and
incorporate ddNTPs efficiently
• Should lack exonuclease activity
• Thermostability required for “cycle
sequencing”
Sanger dideoxy sequencing--basic method
Single stranded DNA
3’
5’
3’
a) Anneal the primer
5’
Sanger dideoxy sequencing: basic method
5’
b) Extend the primer
with DNA polymerase
in the presence of all
four dNTPs, with a
limited amount of a
dideoxy NTP (ddNTP)
Direction of
DNA
polymerase
travel
3’
Sanger dideoxy sequencing: basic method
T
3’
5’
T T
T
3’
ddA
ddA
ddA
ddA
5’
ddATP in the reaction:
anywhere there’s a T in
the template strand,
occasionally a ddA will
be added to the
growing strand
Primer Walking
How to visualize DNA fragments?
• Radioactivity
– Radiolabeled primers (kinase with 32P)
– Radiolabelled dNTPs (gamma 35S or 32P)
• Fluorescence
– ddNTPs chemically synthesized to contain fluoresces
– Each ddNTP fluoresces at a different wavelength
allowing identification
Analysis of sequencing products:
Polyacrylamide gel electrophoresis--good
resolution of fragments differing by a single
dNTP
– Slab gels: as previously described
– Capillary gels: require only a tiny amount of
sample to be loaded, run much faster than
slab gels, best for high throughput
sequencing.
DNA sequencing gels: old school
Different ddNTP used in
separate reactions
Analyze sequencing
products by gel
electrophoresis,
autoradiography
Radioactively labelled primer or dNTP in
sequencing reaction
cycle sequencing: denaturation
occurs during temperature cycles
94°C:DNA denatures
45°C: primer anneals
60-72°C: thermostable DNA
pol extends primer
Repeat 25-35 times
Advantages: don’t need a lot of
template DNA
Disadvantages: DNA pol may
incorporate ddNTPs poorly
An automated sequencer
The output
Current trends in sequencing:
It is rare for labs to do their own sequencing:
--costly, perishable reagents
--time consuming
--success rate varies
Instead most labs send out for sequencing:
--You prepare the DNA (usually plasmid, M13, or PCR product),
supply the primer, company or university sequencing center does the
rest
--The sequence is recorded by an automated sequencer as an
“electropherogram”
Sequencing large pieces of DNA:
the “shotgun” method
• Break DNA into small pieces (typically sizes of around
1000 base pairs is preferable)
• Clone pieces of DNA into M13
• Sequence enough M13 clones to ensure complete
coverage (eg. sequencing a 3 million base pair genome
would require 5x to 10x 3 million base pairs to have a
reliable representation of the genome)
• Assemble genome through overlap analysis using
computer algorithms, also “polish” sequences using
mapping information from individual clones,
characterized genes, and genetic markers
• This process is assisted by robotics
BREAK UP THE GENOME, PUT IT BACK
TOGETHER
~160 kbp
Assemble sequences by matching
overlaps
BAC sequence
~1 kbp
BAC overlaps give genome sequence
Sequence by DNA polymerase -dependent chain extension, one base
at a time in the presence of a reporter (luciferase)
Luciferase is an enzyme that will emit a photon of light in response
to the pyrophosphate (PPi) released upon nucleotide addition by
DNA polymerase
Flashes of light and their intensity are recorded
Height of peak indicates the number of dNTPs
added
This sequence: TTTGGGGTTGCAGTT
Cycle Sequencing
• Cycle sequencing is chain termination
sequencing performed in a thermal cycler.
• Cycle sequencing requires a heat-stable DNA
polymerase.
Fluorescent Dyes
• Fluorescent dyes are multicyclic molecules
that absorb and emit fluorescent light at
specific wavelengths.
• Examples are fluorescein and rhodamine
derivatives.
• For sequencing applications, these molecules
can be covalently attached to nucleotides.
Fluorescent Dyes
• In dye primer sequencing, the primer contains
fluorescent dye–conjugated nucleotides, labeling the
sequencing ladder at the 5′ ends of the chains.
ddA
• In dye terminator sequencing, the fluorescent dye
molecules are covalently attached to the
dideoxynucleotides, labeling the sequencing ladder
at the 3′ ends of the chains.
ddA
Dye Terminator Sequencing
• A distinct dye or “color” is used for each of the four
ddNTP.
• Since the terminating nucleotides can be
distinguished by color, all four reactions can be
performed in a single tube.
A
T
G
T
AC
GT
The fragments are distinguished
by size and “color.”
Dye Terminator Sequencing
The DNA ladder is resolved in one gel lane or in
a capillary.
G
A
T
GA
TC
C
G
T
C
T
G
A
Slab gel
Capillary
Dye Terminator Sequencing
• The DNA ladder is read on an electropherogram.
Slab gel
Capillary
Electropherogram
5′ AGTCTG
Automated Sequencing
• Dye primer or dye terminator sequencing on capillary
instruments.
• Sequence analysis software provides analyzed sequence in
text and electropherogram form.
• Peak patterns reflect mutations or sequence changes.
T/T
5′ AGTCTG
T/A
5′ AG(T/A)CTG
A/A
5′ AGACTG
Alternative Sequencing Methods:
Pyrosequencing
• Pyrosequencing is based on the generation of
light signal through release of pyrophosphate
(PPi) on nucleotide addition.
– DNAn + dNTP  DNAn+1 + PPi
• PPi is used to generate ATP from adenosine
phosphosulfate (APS).
– APS + PPi  ATP
• ATP and luciferase generate light by conversion of
luciferin to oxyluciferin.
Alternative Sequencing Methods:
Pyrosequencing
•
•
•
•
Each nucleotide is added in turn.
Only one of four will generate a light signal.
The remaining nucleotides are removed enzymatically.
The light signal is recorded on a pyrogram.
DNA sequence: A T C A GG CC T
Nucleotide added : A T C A G
C T
Alternative Sequencing Methods:
Bisulfite Sequencing
• Bisulfite sequencing is used to detect
methylation in DNA.
• Bisulfite deaminates cytosine, making uracil.
• Methylated cytosine is not changed by
bisulfite treatment.
• The bisulfite-treated template is then
sequenced.
Bisulfite Sequencing
The sequence of treated and untreated
templates is compared.
Methylated sequence:
GTC
Treated sequence:
GTC
Me
Me
GGC
GGC
Me
Me
GATCTATC
GATUTATC
Me
Me
GTGCA …
GTGUA …
DNA Sequence:
(Untreated) reference: ...GTCGGCGATCTATCGTGCA…
Treated sequence:
...GTCGGCGATUTATCGTGUA…
This sequence indicates that these Cs are methylated.
(we have this)
genome
(we want these)
DNA
“transcriptome”
RNA
“proteome”
protein
DNA microarray -- immobilize many probes
(thousands) in an ordered array, hybridize (base
pair) with labelled mRNA or cDNA
•
•
Generating an array of probes
Identify open reading frames (orfs)
1) PCR each orf (several for each orf), attach (spot)
each PCR product to a solid support in a specific
order (pioneered by Pat Brown’s lab, Stanford)
2) Chemically synthesize orf-specific oligonucleotide
probes directly on microchip (Affymetrix)
A yeast array experiment
vegetative
sporulating
Isolate mRNA
Prepare fluorescently labeled
cDNA with two different-colored
fluors
hybridize
read-out
Example microarray data
Green: mRNA
more abundant in
vegetative cells
Yellow: equivalent
mRNA abundance in
vegetative and
sporulating cells
Red: mRNA more
abundant in
sporulating cells
DNA Microarrays:
An Introduction
Microarray
Result:
Much analysis to
follow
Microarray Technology
The value of DNA microarrays for
studying gene expression
1) Study all transcripts at same time
1) Transcript abundance usually correlates with
level of gene expression--much gene control is at
level of transcription
2)
Changes in transcription patterns often occur as
a response to changing environment--this can be
detected with a microarray
Summary
• Genetic information is stored in the order or sequence of
nucleotides in DNA.
• Chain termination sequencing is the standard method for
the determination of nucleotide sequence.
• Dideoxy-chain termination sequencing has been
facilitated by the development of cycle sequencing and
the use of fluorescent dye detection.
• Alternative methods are used for special applications,
such as pyrosequencing (for resequencing and
polymorphism detection) or bisulfite sequencing (to
analyze methylated DNA).
END Part I