Announcement
No formal Mid-term
However,there will be one test before final,
probably in May,The purpose is to guide your
study and to let you do better in final
Cloning a Disease Gene
There are many ways of cloning a disease
gene,depending on what is known about
the disease,We will show several typical
examples in this part of the lecture
Identification of Hemophilia A (1)
Identification of Hemophilia A (2)
Identification of Hemophilia A (3)
Identification of Hemophilia A (4)
Linkage Analysis
Linkage Map
Generation of Linkage Map and its Genetic
Application
Karyotype,Banding that Characterizes
Chromosmes
Banding at
Different
Resolution
Alternative Way to
Create a Physical
Map,Fingerprinting
How a Whole-Chromosome Sequence is Obtained
Different Levels of DNA Map
If You Can See the Deletion…
If You Cannot See,then You Have to Genetically
Map it
Example,Mapping HD Locus to G8
Fish Signal of G8
Position Cloning Breast Cancer BRCA1(1)
Position Cloning Breast Cancer BRCA1(2),Initial
Mapping BRCA1
Position Cloning Breast Cancer BRCA1(3),Fine
Mapping and Candidate Genes
Sorting out Candidate Genes
Probing Gene Function,Sequence Analysis
Orthologue and Homologue
Other Common Ways to Probe Gene’s Function
Expression Pattern Analysis
Using Cell Culture
Using Model Organisms
Genome Project
Human Genome projects
3 billion nucleotides in human genome were
sequenced
Prepare a entire genome map with overlapping
clones (300-400 kbps per clone = 10,000
clones)
Prepare restriction map of each clone
Sequence each clone
Using overlapping sequences,an entire
sequence of the genome can be constructed.
Or alternatively…
Method 2 for preparing the sequence of a
genome
Prepare overlapping clones of the entire genome (50-200
kbps per clone = 30,000 clones for the human genome)
Take each clone and subclone it into smaller fragments
Sequence each subclone
Using high capacity computer and advanced programs,
the sequence can be constructed from overlapping
fragments.
What has been found from the human
genome project?
About 38,000 genes were found
About 60% of these genes are alternatively spliced
About half of the genome is SINE and LINE elements
About 1-2% of the total genetic material has exon
coding sequences,but combined with introns genes
represent about a quarter of the genome.
The distribution of genes in the genome is highly
uneven,Some chromosomes are gene-rich,other
gene-poor.
Microarray Technology
Why use microarray technology?
Microarray technology gives
researchers the ability to identify
thousands of genes simultaneously to
see how they work in a system rather
than how they work individually.
Application Potential of Microarray technology
Identification of complex genetic diseases
Drug discovery and toxicology
Mutations and polymorphism detection
Pathogen analysis
Differing expression of genes overtime,
between tissues and disease states.
What is a microarray?
A glass or silicon chip spotted with
thousands of pieces of DNA onto
specific locations on the chip.
The DNA is complementary to thousands
of genes with known sequences.
How microarray works?
Oligonucleotide or cDNA sequences representing
genes are laid down on a wafer about the size of a
dime,and each probe represents one gene.
A tissue sample is used to isolate mRNAs,converted
to cDNA and tagged with a fluorescent dye.
The sample is overlaid and allowed to hybridize to the
oligonucleotide/cDNA sequences.
An automated scanner then determines which of the
mRNAs have bound,and the amount of fluorescence
gives and estimate of the amount of gene expression.
The two solutions
Green
fluorescent dye
This is the
experimental
sample
containing
DNA from a cell
expressing
abnormal
behavior or a
cell that is
under
abnormal
conditions.
Red
fluorescent dye
This is the
control sample
containing
DNA from a cell
expressing
normal
behavior under
normal
conditions.
Loading the chip
Analysis of the Results
When the experimental DNA does not
contain the gene in question and the
reference DNA does,a red spot will
appear,In this case the reference
DNA binds to the cDNA and the green
color is washed off leaving only a red
spot.
This indicates that the gene in the
experimental DNA is being down-
regulated relative to the reference
DNA.
Analysis of Results continued...
When the experimental DNA does
contain the gene in question,and the
reference DNA does not,a green spot
will appear,In this case the
experimental DNA binds to the cDNA
and the red color is washed off leaving
only a green spot.
This indicates that the gene in the
experimental DNA is being up-regulated
in relation to the reference DNA.
Analysis of Results continued...
When the gene in question is either
present in both sets of DNA in both sets of
DNA,a yellow spot will appear.
This indicates that the activity of the gene
in the experimental sample has been
unchanged in relation to the reference
sample of DNA.
Analysis of the Results
Problems with the Technique
Can’t identify previously unknown genes.
Cross-hybridization of some sequences
occurs.
Not all mRNA is translated into protein,
therefore samples may not reflect proteins
that are being produced in the cell.
And,it is only an expression!
No formal Mid-term
However,there will be one test before final,
probably in May,The purpose is to guide your
study and to let you do better in final
Cloning a Disease Gene
There are many ways of cloning a disease
gene,depending on what is known about
the disease,We will show several typical
examples in this part of the lecture
Identification of Hemophilia A (1)
Identification of Hemophilia A (2)
Identification of Hemophilia A (3)
Identification of Hemophilia A (4)
Linkage Analysis
Linkage Map
Generation of Linkage Map and its Genetic
Application
Karyotype,Banding that Characterizes
Chromosmes
Banding at
Different
Resolution
Alternative Way to
Create a Physical
Map,Fingerprinting
How a Whole-Chromosome Sequence is Obtained
Different Levels of DNA Map
If You Can See the Deletion…
If You Cannot See,then You Have to Genetically
Map it
Example,Mapping HD Locus to G8
Fish Signal of G8
Position Cloning Breast Cancer BRCA1(1)
Position Cloning Breast Cancer BRCA1(2),Initial
Mapping BRCA1
Position Cloning Breast Cancer BRCA1(3),Fine
Mapping and Candidate Genes
Sorting out Candidate Genes
Probing Gene Function,Sequence Analysis
Orthologue and Homologue
Other Common Ways to Probe Gene’s Function
Expression Pattern Analysis
Using Cell Culture
Using Model Organisms
Genome Project
Human Genome projects
3 billion nucleotides in human genome were
sequenced
Prepare a entire genome map with overlapping
clones (300-400 kbps per clone = 10,000
clones)
Prepare restriction map of each clone
Sequence each clone
Using overlapping sequences,an entire
sequence of the genome can be constructed.
Or alternatively…
Method 2 for preparing the sequence of a
genome
Prepare overlapping clones of the entire genome (50-200
kbps per clone = 30,000 clones for the human genome)
Take each clone and subclone it into smaller fragments
Sequence each subclone
Using high capacity computer and advanced programs,
the sequence can be constructed from overlapping
fragments.
What has been found from the human
genome project?
About 38,000 genes were found
About 60% of these genes are alternatively spliced
About half of the genome is SINE and LINE elements
About 1-2% of the total genetic material has exon
coding sequences,but combined with introns genes
represent about a quarter of the genome.
The distribution of genes in the genome is highly
uneven,Some chromosomes are gene-rich,other
gene-poor.
Microarray Technology
Why use microarray technology?
Microarray technology gives
researchers the ability to identify
thousands of genes simultaneously to
see how they work in a system rather
than how they work individually.
Application Potential of Microarray technology
Identification of complex genetic diseases
Drug discovery and toxicology
Mutations and polymorphism detection
Pathogen analysis
Differing expression of genes overtime,
between tissues and disease states.
What is a microarray?
A glass or silicon chip spotted with
thousands of pieces of DNA onto
specific locations on the chip.
The DNA is complementary to thousands
of genes with known sequences.
How microarray works?
Oligonucleotide or cDNA sequences representing
genes are laid down on a wafer about the size of a
dime,and each probe represents one gene.
A tissue sample is used to isolate mRNAs,converted
to cDNA and tagged with a fluorescent dye.
The sample is overlaid and allowed to hybridize to the
oligonucleotide/cDNA sequences.
An automated scanner then determines which of the
mRNAs have bound,and the amount of fluorescence
gives and estimate of the amount of gene expression.
The two solutions
Green
fluorescent dye
This is the
experimental
sample
containing
DNA from a cell
expressing
abnormal
behavior or a
cell that is
under
abnormal
conditions.
Red
fluorescent dye
This is the
control sample
containing
DNA from a cell
expressing
normal
behavior under
normal
conditions.
Loading the chip
Analysis of the Results
When the experimental DNA does not
contain the gene in question and the
reference DNA does,a red spot will
appear,In this case the reference
DNA binds to the cDNA and the green
color is washed off leaving only a red
spot.
This indicates that the gene in the
experimental DNA is being down-
regulated relative to the reference
DNA.
Analysis of Results continued...
When the experimental DNA does
contain the gene in question,and the
reference DNA does not,a green spot
will appear,In this case the
experimental DNA binds to the cDNA
and the red color is washed off leaving
only a green spot.
This indicates that the gene in the
experimental DNA is being up-regulated
in relation to the reference DNA.
Analysis of Results continued...
When the gene in question is either
present in both sets of DNA in both sets of
DNA,a yellow spot will appear.
This indicates that the activity of the gene
in the experimental sample has been
unchanged in relation to the reference
sample of DNA.
Analysis of the Results
Problems with the Technique
Can’t identify previously unknown genes.
Cross-hybridization of some sequences
occurs.
Not all mRNA is translated into protein,
therefore samples may not reflect proteins
that are being produced in the cell.
And,it is only an expression!
