Lecture 4 PHBC731

Molecular Biology Techniques

Part 2

Mohamed Zakaria Gad

Prof. of Biochemistry
Mohamed.gad@guc.edu.eg
1
 DNA Sequencing
In 1963, F. Sanger of Britain
developed sequencing procedure Frederick
for DNA. Sanger
winner of two
Nobel Prizes!

Application: Analysis of isolated and

recombinant DNA molecules
Two methods are used: Maxam-Gilbert method
and the Sanger method. Both depend on an initial
fractionation of the DNA into small pieces.
2
Sanger’s Method
DNA synthesis occurs in presence of chemically modified (dideoxynucleotides)
radiolabeled nucleotides.
nucleotides Four incubations are set up in 4 test tubes as follows:

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*

Incorporation of radiolabeled nucleotides in the newly synthesized DNA

complementary sequences terminate the DNA synthesis. This creates in the 4
test 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 per3 day
can be sequenced.
4
 DNA Microarray Method
“also known as gene or genome chip, DNA chip, or
gene
It isarray”
a collection of microscopic DNA spots,
commonly representing single genes, arrayed on a
solid surface by covalent attachment to chemically
suitable matrices. Qualitative or quantitative
measurements with DNA microarrays utilize the
selective nature of DNA-DNA or DNA-RNA
hybridization under high-stringency conditions and
fluorophore-based detection.
Application: Studying which genes are active
and which are inactive in different cell types
5
What is DNA Microarray Technology?
Although all of the cells in the human body contain
identical 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 various
genes do not perform properly.

This helps researchers to learn more about many

different diseases, including heart disease, mental
illness, infectious diseases and cancer

6
How does DNA microarray technology work ?

Researchers have a
database of over 40,000
gene sequences that they
can use for this purpose.
When a gene is
activated, the mRNA
produced by the cell is
complementary, and
therefore will bind to the DNA microarrays are created by
original portion of the robotic machines that arrange
minuscule amounts of hundreds or
DNA strand from which it thousands of gene sequences on a
was copied. single microscope slide.
7
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,

active it produces many molecules
of mRNA, which hybridize to the DNA on the microarray and
generate a very bright fluorescent area. Genes that are
somewhat 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. 8
START FLASH MOVIE ON
DNA MICROARRAY

chip.swf

9
TUMOR
MARKERS

10
What is a tumour marker ??

Any substance that can be related to the presence

or 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.

11
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.

12
What are the uses of
tumour markers ??

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.

13
Type Use of Tumour Marker
Monitoring Decline in conc. of tumour marker is an indication of
treatment the success of the treatment.
Assessing It is valuable to continue to monitor the tumour
follow-up markers, 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 confirm
the 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 in
choriocarcinoma.
Screening In routine clinical practice tumour markers should
not be used to screen for malignancy. The
exception is the screening of specific high-risk
14
populations.
Clinical situations where tumour markers have been found to be useful
Marker Tumour Screenin Diagnos Prognosi Monitori Follow-
g is s ng up
AFP Germ cell    
AFP Hepatoma    
HCG Germ cell    
HCG Choriocarcinoma     
CA 125 Ovarian   
Acid Prostate   
phosphata
se
PSA Prostate   
CEA Colorectal  

Calcitonin Medullary carcinoma of    

thyroid
Paraprotei Myeloma   
AFP=alpha- fetoprotein, HCG=Human Chorionic Gonadotrophin,CA-125=cancer antigen-125,
n 15
PSA=prostate-specific antigen, CEA=carcinoembryonic antigen.
WHO ARE WE ?
 WHAT WE KNOW
ABOUT OUR GENETIC
MAKEUP

17
By Numbers
 The human genome contains 3 billion nucleotide
bases (A, C, T & G). 
 The average gene consists of 3000 bases, but
sizes vary greatly, with the largest known human
gene being dystrophin at 2.4 million bases.
 The 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 the
same in all people.
 The functions are unknown for over 50% of
18
discovered genes.
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 and G bases
repeating over and over often occur adjacent
to gene-rich areas. These CpG islands are
believed to help regulate gene activity.
Chromosome 1 has the most genes (2968),
19
and the Y chromosome has the fewest (231).
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 the 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%),
20
and the fly (3%).
Variations and Mutations
 Scientists have identified about 3 million locations
where single-base DNA differences (SNPs) occur
in 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
21
eggs.
 Striking Facts
 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 your
parents, 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 and
gorillas.
 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 22
they interact with each other and with our
X WHAT WE STILL DO NOT KNOW ?
Organizatio Structure Regulatio Location
n n s
Chromosomes Genes

Number Function
s

Amount Functio
Type
n Conte
s
nt
Function Proteomes
Non-coding DNA

Informatio Distributi
n Conservation Multigene
on
disease

Gene sequence
Health
SNPs

Evolutio Disease
Disease
n susceptibility 24
 Any Questions?

“The important thing is not to stop questioning"

•
Einstein

25
 ‫كلمات باقية‬

‫دقات قلب المرء قائلة له إن الحياة دقائق و ثوان‬

‫فإرفع لنفسك بعد موتك ذكرهافالذكر لإلنسان عمر ثان‬

‫من أجمال ابيات شوقي في‬

‫رثاء مصطفي كامل‬

‫‪26‬‬
27