Molecular Biology Techniques (Part 2)

7/30/2019 Molecular Biology Techniques (Part 2)

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Lecture 4 PHBC731

Mohamed Zakaria Gad

Prof. of Biochemistry

[email protected]

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!
http://www.rsc.org/images/FEATURE-sanger1-250_tcm18-42175.jpg


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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*


+ radioactive A*


+ radioactive G*


+ 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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X WHAT WE STILL

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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Molecular Biology Techniques (Part 2)