molecular biology techniques (part 2)
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Lecture 4 PHBC731
Mohamed Zakaria Gad
Prof. of Biochemistry
Molecular Biology Techniques
Part 2
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DNA Sequencing
Application: Analysis of isolated and
recombinant DNA molecules
Two methods are used: Maxam-Gilbert method and theSanger method. Both depend on an initial fractionation
of the DNA into small pieces.
In 1963, F. Sanger of Britaindeveloped sequencing procedure for
DNA.
Frederick
Sanger
winner of two
Nobel Prizes!
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Sangers MethodDNA synthesis occurs in presence ofchemically modified (dideoxynucleotides)
radiolabeled nucleotides. Four incubations are set up in 4 test tubes as follows:
Incorporation of radiolabeled nucleotides in the newly synthesized DNA
complementary sequences terminate the DNA synthesis. This creates in the 4test tubes complementary DNA sequences with different lengths. By
electrophoresis based on molecular size, the sequence of nucleotides can be
read directly from the gel after detection of the radioactive sequences only by
autoradiography.
Sanger method has now been automated so that thousands of bases per daycan be sequenced.
Tube1: DNA strand + DNA polymerase + primer + nonradioactive A,T,G,C
+ radioactive T*
Tube2: DNA strand + DNA polymerase + primer + nonradioactive A,T,G,C
+ radioactive A*
Tube3: DNA strand + DNA polymerase + primer + nonradioactive A,T,G,C
+ radioactive G*
Tube4: DNA strand + DNA polymerase + primer + nonradioactive A,T,G,C
+ radioactive C*
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http://localhost/var/www/apps/conversion/%D9%85%D8%AD%D8%A7%D8%B6%D8%B1%D8%A7%D8%AA%20%D8%A7%D9%84%D9%83%D9%8A%D9%85%D9%8A%D8%A7%D8%A1%20%D8%A7%D9%84%D8%AD%D9%8A%D9%88%D9%8A%D8%A9/Molecular%20Biology/STUDENTS/animations/beyondhgp.pps -
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DNA Microarray Method
Application: Studying which genes are active
and which are inactive in different cell types
It is a collection of microscopic DNA spots, commonlyrepresenting single genes, arrayed on a solid surface by covalentattachment to chemically suitable matrices. Qualitative orquantitative measurements with DNA microarrays utilize theselective nature of DNA-DNA or DNA-RNA hybridizationunder high-stringency conditions and fluorophore-baseddetection.
also known as gene or genome chip, DNA chip, or gene array
http://images.google.com.eg/imgres?imgurl=http://radio.weblogs.com/0105910/images/ecoli_dna.jpg&imgrefurl=http://radio.weblogs.com/0105910/2004/02/15.html&h=224&w=300&sz=29&tbnid=o-sO7x9gGFupPM:&tbnh=87&tbnw=116&prev=/images%3Fq%3Ddna%2Bmicroarray%26um%3D1&start=3&sa=X&oi=images&ct=image&cd=3 -
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Although all of the cells in the human body containidentical genetic material, the same genes are not
active in every cell. Studying which genes are active
and which are inactive in different cell types helps
scientists to understand both how these cells function
normally and how they are affected when variousgenes do not perform properly.
This helps researchers to learn more about many
different diseases, including heart disease, mentalillness, infectious diseases and cancer
What is DNA Microarray Technology?
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Researchers have a
database of over 40,000
gene sequences that they
can use for this purpose.
When a gene isactivated, the mRNA
produced by the cell is
complementary, and
therefore will bind to theoriginal portion of the
DNA strand from which it
was copied.
How does DNA microarray technology work ?
DNA microarrays are created by
robotic machines that arrange
minuscule amounts of hundreds or
thousands of gene sequences on a
single microscope slide.
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To determine which genes are turned on and which are turned
off in a given cell:
1) Collect the mRNA molecules present in that cell.
2) label each mRNA molecule by attaching a fluorescent dye.
3) Place the labeled mRNA onto a DNA microarray slide. The
mRNA that present in the cell will then hybridize - or bind - to
its complementary DNA on the microarray.
4) use a special scanner to measure the fluorescent areas on
the microarray.
If a particular gene is very active, it produces many molecules
of mRNA, which hybridize to the DNA on the microarray and
generate a very bright fluorescent area. Genes that aresomewhat active produce fewer mRNAs, which results in
dimmer fluorescent spots. If there is no fluorescence, none
of the messenger molecules have hybridized to the DNA,
indicating that the gene is inactive.
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START FLASH MOVIE ON
DNA MICROARRAY
chip.swf
http://localhost/var/www/apps/conversion/tmp/scratch_3/chip.swfhttp://localhost/var/www/apps/conversion/tmp/scratch_3/chip.swf -
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TUMOR MARKERS
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What is a tumour marker ??
Any substance that can be related to the presenceor progress of a tumour.
In practice, clinical biochemists usually measure
these markers in blood.
A tumour marker in plasma has been secreted or
released by the tumour cells. Such markers are not
necessarily unique products of the malignant cells,
but may simply be expressed by the tumour in a
greater amount than by the normal cells.
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How are tumour markers classified ??
Could be:Hormones, e.g. human chorionic gonadotrophin
(HCG) secreted by choriocarcinoma.
Enzymes, e.g. prostatic specific antigen (PSA) in
prostate carcinoma.
Antigens, e.g. carcinoembryonic antigen (CEA) in
colorectal carcinoma.
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They are of most
value in monitoring
treatment and
assessing follow-up as shown in fig.,
but are also used
in diagnosis and
screening for the
presence of the
disease.
What are the uses of
tumour markers ??
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Type Use of Tumour Marker
Monitoring
treatment
Decline in conc. of tumour marker is an indication of
the success of the treatment.
Assessingfollow-up
It is valuable to continue to monitor the tumourmarkers, long after the stabilization with treatment.
An increase indicates recurrence of the malignancy.
Diagnosis Markers alone are rarely used to establish a
diagnosis. Their detection in blood will often confirmthe diagnosis.
Prognosis To be of value in prognosis, the conc. of the marker
in plasma should correlate with the tumour mass
e.g. HCG correlates well with the tumour mass inchoriocarcinoma.
Screening In routine clinical practice tumour markers should
not be used to screen for malignancy. The
exception is the screening of specific high-risk
populations.
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Clinical situations where tumour markers have been found to be useful
Marker Tumour Screening Diagnosis Prognosis Monitoring Follow-
up
AFP Germ cell l l l l
AFP Hepatoma l l l l
HCG Germ cell l l l l
HCG Choriocarcinoma l l l l l
CA 125 Ovarian l l l
Acid
phosphatase
Prostate l l l
PSA Prostate l l l
CEA Colorectal l l
Calcitonin Medullary carcinomaof thyroid
l l l l
Paraprotei
n
Myeloma l l l
AFP=alpha- fetoprotein, HCG=Human Chorionic Gonadotrophin, CA-125=cancer antigen-125,
PSA=prostate-specific antigen, CEA=carcinoembryonic antigen
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WHO ARE WE ?
http://www.stjulies.org/Carol%20Bautista%20PROFILE.htm -
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WHAT WE KNOW ABOUT
OUR GENETIC MAKEUP
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By Numbers
Human genome contains 3 billion nucleotide bases
(A, C, T & G).
Average gene consists of 3000 bases, but sizes
vary greatly, with the largest known human gene
being dystrophin at 2.4 million bases.
Total no. of genes is estimated at ~ 30,000--much
lower than previous estimates of 80,000 - 140,000.
Almost all (99.9%) nucleotide bases are exactly thesame in all people.
The functions are unknown for over 50% of
discovered genes.
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How It's Arranged
Genes appear to be concentrated in random
areas along the genome, with vast expanses
of noncoding DNA between.
Stretches of up to 30,000 C & G bases
repeating over and over often occur adjacent
to gene-rich areas. These CG islands are
believed to help regulate gene activity.
Chromosome 1 has the most genes (2968),
and Y chromosome has the fewest.(231).
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Intervening-Sequences ?
Less than 2% of the genome codes for proteins.
Repeated sequences that do not code for proteins
make up at least 50% of human genome.
Repetitive sequences are thought to have no
direct functions, but they shed light on
chromosome structure and dynamics. Over time,
these repeats reshape the genome by rearranging
it, creating entirely new genes, and modifying and
reshuffling existing genes.
The human genome has a much greater portion
(50%) of repeat sequences than the worm (7%),
and the fly (3%).
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Variations and Mutations
Scientists have identified ~ 3 million locations
where single-base DNA differences (SNPs) occurin humans. This information promises to
revolutionize the processes of finding chromosomal
locations for disease-associated sequences and
tracing human history.
The ratio of germline (sperm or egg cell) mutations
is 2:1 in males vs females. Researchers point to
several reasons, including the greater no. of cell
divisions required for sperm formation than for
eggs.
St iki F t
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Modern man comes from the group Homo sapiens,
which merged from Africa around 150,000 years ago.
Genes are useless by themselves and the proteins
they produce do all the work.
Written out in full, the genome found in every cell of
our body would fill an average-size book, 600,000
pages long.
You share ~ 50% of your genes with each of yourparents, children, brothers & sisters., 25% of your
genes with all your grandparents, uncles & aunts,
12.5% with your cousins.
Our genes is 98.5% identical to those of chimpanzees;
closer genetically than those between chimps andgorillas.
We think we are very smart we share 50% of our
genes with worm, 30% with banana. So, not the no. of
genes which gives us our complexity, but the way they
interact with each other and with our environment.
Striking Facts
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DO NOT KNOW ?
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Genes
Number
Locations
Functions
Regulation
Chromosomes
StructureOrganization
Non-coding DNA
TypesAmount
DistributionInformation
Function
SNPsHealth
Disease
Proteomes
ContentFunction
Gene sequence
Conservation
Evolution Disease
susceptibility
Multigene
disease
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Any Questions?
The important thing is not to stop questioning"
Einstein
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