Human Genome: sequence, structure, diseases

1
Human Genome sequence, structure, diseases

• Lecture 8
• BINF 7580
• Fall 2005

2
Every cell of the body contains a full set of
chromosomes and identical genes (with only a few
exceptions). However only a fraction of these
genes are turned on, and it is the subset that
is "expressed" that confers unique properties to
each cell type. Gene expression" is the term
used to describe the transcription of the
information contained within the DNA, into mRNA
molecules. Scientists study the kinds and amounts
of mRNA produced by a cell to learn which genes
are expressed, and how the cell responds to its
changing needs.
3
The dream of biologists to investigate the
behavior of cells at any given moment. The state
of a cell at any given time is governed by which
subset of its genes is expressed at a given time.

Recall the central dogma genes are transcribed
into mRNA and a complete collection of mRNAs,
including alternatively spliced ones, is called
the organisms transcriptome.
Idea measure the amount of mRNA to see which
genes are being expressed in (used by) the
cell. Measuring protein might be better, but is
currently harder.
4
Today lecture is about DNA microarrays Scientists
use DNA microarrays to measure the expression
levels of large numbers of genes simultaneously.
Why Learn about Microarrays ?
Microarray test allows one to answer the
fundamental question of molecular biology and
Genetics How are genes expressed?
We know that all cells contain the same genetic
information, but skin cells are different from
liver cells or kidney cells or brain cells.

? These differences come about because different
genes are expressed at high levels in different
cells.
5
In pre-genomic era the dominant was the
investigation of a single genes. now we are able
to investigate a snapshot of all of the genes
expressed in a cell at a given time.
One way to obtain usefull information about
genome is to determine which genes are induced or
repressed in the given cell in
a response to a phase of
the cell cycle or a response to the enviroment,
such a treatment with a hormone.
A pattern of gene expression may also be an
indicator of abnormal cellular regulation and is
a useful toll in diseases diagnostics
6

• Microarray
• Extremely useful and powerful technology given
  a sample of human tissue, allows you to determine
  the expression level of all human genes within
  that tissue.
• Now extremely widely used, not only in research
  laboratories but within commercial companies and
  diagnostically in hospitals.

7
Some areas in which microarrays play a major
role gene regulation development
evolution disease One common use of DNA arrays
is to determine which genes are activated and
which genes are repressed when two populations of
cells are compared.
All genes are
measured simultaneously. In medicine, DNA arrays
already play an important role in therapy by
observing drug response at a molecular level.
In nutrition, DNA arrays and
appropriately designed studies could help design
diet and possible nutritional supplements. This
is leading towards individualized treatments.
8
If we could count the number of mRNA molecules
from each gene in a single cell at a particular
time, we might get this An expression profile ?
Question How do we make these measurements?
9
The main principle - How do microarrays work? The
biological principle complementarities
sequences of DNA or RNA molecules containing
complementary base pairs have a natural tendency
to bind together. ...A
A G C A G T C G A T G C T A G...
.


...T T C G T C A G C T A C G A T C... If we know
the target mRNA sequence we can build a probe for
it using the complementary sequence. The probe
location tells us about the gene.
10
a microarray experiment grow or provide two
different populations of cells isolate cells
from the solution (if necessary) extract mRNA
from the cells, RNA will be floating in the
extraction buffer remove RNA and place in fresh
tube
I population of cells II population of
cells
11
a microarray experiment
make complementary DNA (cDNA) using reverse
transcriptase enzyme for both samples
Note the idea of transforming mRNA into cDNA
was suggested for gene detection the expressed
segments of DNA are converted into fragments of
cDNA these fragments are called
expressed sequence tags
(ESTs) these ESTs can be used for assembling
expressed genes
12
Method for preparing cDNA from a
mRNA Twenty-nucleotide-long oligo(dT) primers
hybridize to the poly(A) tail of mRNA. Enzyme
(reverse transcriptase) are used to synthesize a
DNA copy. Ribonuclease H is then used to
specifically degrade most of the RNA component of
the RNA-DNA hybrid. Some remaining portions of
the original RNA are then used as primers for DNA
polymerase. The resulting double stranded DNA
can be cloned into a vector and maintained as a
cDNA library.
13
a microarray experiment
color each cDNA sample (one with green and the
other with red dye) mix two cDNA samples in one
tube
Question Think, why do we need labeling ?
14
provide the chip (microarray) where each spot is
made of DNA that can base pair with cDNA
The idea of a microarray array" as "to place in
an orderly arrangement".
It is important that the gene
sequences in a microarray are attached to their
support (glass slides, or silicon chips or
nylon membranes) in an orderly or fixed way.
Why?
A spot size may be as small as 150 µ in diameter
A grid is typically a few centimeters across.
15
The probes are physically spotted onto particular
positions on a support using a robot and
immobilized using specific surface chemistry.
because a researcher uses the location of each
spot in the array to identify a particular gene
sequence.
The spots themselves can be DNA, cDNA, or
oligonucleotides (5 to 50 nucleotides long). is
simply to lay down a field of thousands of these
probes in perhaps a 5 sq cm area.
16
a microarray experiment incubate mixed cDNA
with DNA chip wash off unbound cDNA and then
see whats bound to the microarray
After the array is exposed to the mixture, each
element of the array to which some component of
the transcript has become attached bears the
radioactive or fluorescent tag.
17
place microarray in a darkened box and scan
with green and then red laser in order to detect
cDNA hybridization
red green yellow
A typical microarray on a glass slide (made from
two samples) looks like
Hybridization to array ?
18
Once the microarray is constructed, the target
mRNA population is labeled, typically with a
fluorescent dye, so that hybridization to the
probe spot can be detected when scanned with a
laser. The image is the data. Trivially, if
fluorescence is observed for a gene in one
population but not another, the gene can be
inferred to be on or off, respectively. For
example
19
Consider two cells cell type 1, a healthy cell,
and
. cell type 2,
a diseased cell. Both contain an identical set
of four genes, A, B, C, and D. The goal of
Microarray experiment in determining the
expression profile of these four genes in the two
cell types. To do this, we 1)
isolate mRNA from each cell type

2) use this mRNA as templates to generate cDNA
with a "fluorescent tag" attached. Different tags
(red and green) are used so that the samples can
be differentiated in subsequent steps.
3) the two labeled samples
are then mixed and incubated with a microarray
containing the immobilized genes A, B, C, and D.
The labeled molecules bind to the sites on the
array corresponding to the genes expressed in
each cell.
20
4) After this hybridization step is complete, we
place the microarray in a "reader" or "scanner"
that consists of some lasers, a special
microscope, and a camera. The fluorescent tags
are excited by the laser, and the microscope and
camera work together to create a digital image of
the array.
GREEN represents Control DNA, where either DNA or
cDNA derived from normal tissue is hybridized to
the target DNA.
RED represents Sample DNA, where either DNA or
cDNA is derived from diseased tissue hybridized
to the target DNA.
 YELLOW represents a combination of Control and
Sample DNA, where both hybridized equally to the
target DNA. BLACK represents areas where neither
the Control nor Sample DNA hybridized to the
target DNA.
21
5) These data are then stored in a computer, and
a special program is used to calculate the
red-to-green fluorescence ratio.

The program then
creates a table that contains the ratios of the
intensity of red-to-green fluorescence for every
spot on the array. Each spot on an array is
associated with a particular gene. Each color in
an array represents either healthy (control) or
diseased (sample) tissue.

Some microarray experiments can
contain up to 30,000 target spots. Thus this
data show whether the gene, or mutation, is
present in either the control and/or sample DNA.
It will also provide an estimate of the
expression level of the gene(s) in the sample and
control DNA. For example, both cell types express
gene A at the same level, the cell 1 expresses
more of gene B, the cell 2 expresses more of gene
C, and that neither cell expresses gene D.
22
Analysis of Microarray data overexpressed gene.
If a gene is overexpressed in a certain disease
state, then more sample cDNA, as compared to
control cDNA, will hybridize to the spot
representing that expressed gene

(so, in
turn, the spot will fluoresce red with greater
intensity than it will fluoresce green. If this
is the case, treatment appropriate for that
disease can be initiated.

23
expression patterns. As researchers use
expression chips to detect expression patternsto
examine changes in gene expression over a given
period of time, such as within the cell cycle.
Analysis of the collected data could further
elucidate details of the cell cycle and its
"clock", providing much needed data on the points
at which gene mutation leads to cancerous growth
as well as sources of therapeutic
intervention. new drugs. Expression chips can be
used to develop new drugs. For instance, if a
certain gene is overexpressed in a particular
form of cancer, researchers can use expression
chips to see if a new drug will reduce
overexpression and force the cancer into
remission.
24
disease diagnosis. Expression chips could also
be used in disease diagnosis as well, e.g., in
the identification of new genes involved in
environmentally triggered diseases, such as those
diseases affecting the immune, nervous, and
pulmonary/respiratory systems
25
Problems in Microarray experiments Gene
expression profiling experiments 1) Differences
among samples for example, two strains of E.
Coli cells can be made isogenic (identical
genetically), except for one gene. the set of
experimental conditions must be standardized in
order to compare these experiments. 2) RNA
isolation procedure the largest source of error
in DNA array data comes from mRNA expression
levels in different cell populations even when
care is taken to culture cells under identical
conditions.
26
3) Rapid turnover of mRNA in bacterial cells.
mRNA has a turnout rate from a few seconds to
several minutes, while in eukaryotes it usually
takes several hours or days 4) Normalization
methods - data sets must be comparable the
problem arises from the fact that cells cultured
under different conditions may have significantly
different amounts of total RNA important
considerations include as well

the number of cells in the sample,

total RNA isolation
efficiency,
mRNA
isolation and labeling efficiency,

hybridization efficiency,

signal measurement sensitivity
27

• Why Are Microarrays Important?
• There are two reasons at least
• Microarrays may contain a very large number of
  genes and give possibility to examine the
  expression of hundreds or thousands of genes at
  once.
• Microarray has a small size and useful when the
  sample to be studied is small.

Microarrays may be used to assay gene expression
within a single sample or to compare gene
expression in two different cell types or tissue
samples, such as in healthy and diseased tissue.
28

• Let us look at detecting differences between
  acute lymphoblastic
  leukemia (ALL) and
  acute myeloid leukemia
  (AML) .
• This was one of the first successful uses of
  microarrays in cancer classification.
• T.R. Golub et. al (1999) Molecular Classification
  of Cancer Class Discovery and Class Prediction
  by Gene Expression Monitoring. Science, vol. 286,
  531-537.
• Total mRNA was extracted from bone marrow taken
  from patients with ALL and AML.
• Converted to cDNA by reverse transcription
  (since mRNA is
  sensitive to degradation).

29
Spot Interpretation Green Spot higher
expression in ALL. Red Spot higher expression
in AML. Yellow Spot equal expression in
both. Black Spot not expressed in either.
AML cDNAs
ALL cDNAs
Cy3 DyeLabel
Cy5 DyeLabel
Mix and hybridize onto slide
30
Image Analysis
To detect genes which are expressed differently
between two samples. For example, detecting those
genes expressed differently between ALL and AML,
which may explain differences between these types
of leukemia.