Biotechnology and Genetic Engineering-PBIO 450/550

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14 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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Chapter 9
-
Molecular Diagnostics


Enzyme
-
Linked Immunosorbent Assay (ELISA)


Monoclonal Antibodies


DNA Diagnostic Systems (DNA fingerprinting)


Molecular Diagnosis of Genetic Disease

Old vs. New Molecular Diagnostics


Old: grow cells/pathogen
-
>test


Such growth can be a problem as it is
sometimes slow, costly, and specific


New: direct test (either immunological or DNA
based)


Detection must be: specific, sensitive, and
simple (fast and automatable are also nice)

Fig. 9.1 Enzyme
-
Linked Immunosorbent Assay
(ELISA): immunological detection

Target molecule

antigenic site

i i i i i i i i i i i i i i i

Support

A. Bind sample to the support (commonly plastic or a membrane)

B. Add primary antibody; wash

C. Add secondary antibody
-
enzyme conjugate; wash

D. Add substrate

Y

Y

Y

Y

bound primary

antibody

Y

E

Y

E

Y

E

Y

E

enzyme linked

secondary antibody

colorless substrate

colored product

Fig. 9.2 Target antigens and polyclonal versus
monoclonal antibodies

Polyclonal antibodies

are made against and react with

multiple antigenic sites (epitopes) on a target antigen.

Monoclonal antibodies
are directed against a particular

antigenic site.

Target antigen

with various antigenic

determinants (epitopes)

1

2

3

4

5

6

7

Fig. 9.4 Procedure for
producing a monoclonal
antibody to protein X


Note: B lymphocytes or B cells produce
antibodies but do not reproduce in
culture. Some B cells can become
cancerous and are known as myelomas
which can reproduce in culture.


See
http://bcs.whfreeman.com/lodish5e/pages/bcs
-
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|09000|10000|11000|12000|13000|14000|15000|16000
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Fig. 9.3 Explanation of how HAT medium works

Myeloma cells are HGPRT
-

and will die on
HAT

media having
h
ypoxanthine,

a
minopterin (an antifolate), and
t
hymidine.

Spleen cells are HGPRT
+

, so spleen
-
myeloma (hybridoma) cells can grow on HAT.

(Note: spleen cells by themselves cannot grow in culture.)

Fig. 9.5 Targets for diagnostic monoclonal antibodies


Polypeptide hormones (chorionic
gonadotropin, growth hormone)


Tumor markers (Prostate
-
specific antigen)


Cytokines (interleukins 1
-
8)


Drug monitoring (cyclosporin)


Miscellaneous targets (Vitamin B
12
)


Infectious diseases (Chlamydia, Herpes,
Rubella, Hepatitis B, Legionella, HIV)

Fig. 9.6 DNA diagnostic systems

1.
Bind ssDNA (target) to membrane

2.
Hybridize to labeled ssDNA or RNA (probe)

3.
Wash membrane to remove unbound probe

4.
Detect hybrid sequences formed between the
probe and target DNA (concern: false +s &
-
s)

membrane

DNA based diagnosis of Malaria and
Typanosoma

cruzi


A DNA probe from a highly repeated DNA sequence
of
Plasmodium falciparum
, the parasite that causes
malaria, is used to screen blood samples via
hybridization assays


DNA primers are made against the ends of a 188 bp
repeated sequence contained in the protozoan
parasite
Typanosoma cruzi
, the causative agent of
Chagas disease and used in a PCR/polyacrylamide gel
electrophoresis detection method


Other examples of DNA
-
based detection:
Salmonella
typhi

(food poisoning), certain
E. coli

(gastroenteritis),
Mycobacterium tuberculosis

(tuberculosis), etc.

Nonradioactive Hybridization Procedures


Use of biotin
-
labeled nucleotides in DNA probes
instead of
32
P, then add avidin (streptavidin) which
binds to biotin, and then add biotin attached to an
enzyme like alkaline phosphatase for detection (see
Fig. 9.11)


Note that fluorescent dyes can also be attached to
DNA primers for detecting amplified DNA products
(see Fig. 9.12)


Nonradioactive Hybridization Procedures

Fig. 9.9 Nonradioactive Hybridization
Procedures: Molecular Beacons

Target DNA

.

Molecular beacon probe

Hybridization

Fluorophore

Quencher

Fluorescence!!!

(No Fluorescence)

DNA Fingerprinting & Forensics


History


Uses of DNA Profiling


Hypervariable DNA sequences examined (RFLPs, VNTRs,
STRs, SNPs, mitochondrial DNA, Y chromosomal DNA)


Methods (Southerns & PCR)


Statistical considerations


Technical considerations


Databases and Privacy

DNA Fingerprinting


You're 99.9% identical


But of course, you are unique
--
in a genome of three
billion letters, even a 0.1 % difference translates into
three million differences.


These differences (or polymorphisms) reside in
several places in the genome, often in microsatellites


Examples of such polymorphisms include VNTRs,
STRs, RFLPs and SNPs


DNA Fingerprinting



Focuses on the 0.1
-
1.0% of human DNA that is
unique


First described in 1985 by Dr. Alec Jeffreys in England


DNA evidence is admissible in courts


Labs such as Cellmark Diagnostics and Lifecodes
Corporation are examples of companies which
provide such DNA evidence to courts, but states and
many U.S. cities have labs for DNA fingerprinting


Have any of you worked in a crime lab?


Uses of DNA fingerprinting


Paternity testing


Identification of criminals (e.g. murderers, rapists,
letter bombers)


Immigration disputes (family relationships)


Identification of deceased individuals with mutilated
or decomposed bodies (e.g
., the military, 9/11 victims)


Identifying the sperm donor who “decorated” Monica
Lewinsky’s blue dress


How is DNA fingerprinting done?


DNA obtained from hair, semen, blood, sweat, saliva,
bone or any other tissue (often found at a crime scene)


Can be done by southern blotting with an appropriate
probe or by a PCR method using appropriate primers


Can use single locus probes/primers or multilocus
probes/primers


DNA can be resolved on a gel or by a capillary
electrophoresis system

Sequences examined in DNA fingerprinting


VNTRs
-
variable number tandem repeats; composed of 8
-
80 bp repeat units (e.g., [GCGCAATG]n) which are
tandemly repeated so that the overall length is 1
-
30 kb


STRs
-
short tandem repeats; composed of 2
-
7 bp repeat
units (e.g., [AC]n) which are tandemly repeated so that
the overall length is less than 1 kb


RFLPs
-
restriction fragment length polymorphisms


SNPs
-
single nucleotide polymorphisms


Mitochondrial DNA
-
maternal inheritance, tends to be
more stable than nuclear DNA


Y chromosome DNA
-

passed from father to son

DNA fingerprinting: an example


D1S80, a VNTR located on human chromosome 1,
contains a 16 bp repeat unit


The number of repeats varies from one individual to the
next, and is known to range from 14
-
41


Some examples of DNA fingerprinting


Paternity cases


Crime scenes

Determining the probability of a match



Relies on statistics


Analysis depends upon your ethic background
(i.e. African American, Caucasian, Hispanic
Asian, etc.)

Technical Considerations



Preserve the integrity of DNA sample


Avoid DNA contamination & degradation


Avoid incomplete digestions if REs are used


Use standard hybridization conditions


Use standard PCR primers and procedures


Gel analysis is less reproducible than capillary
electrophoresis of PCR products


Difficulties in interpreting bands on a gel or X
-
ray film

DNA databases


Already in place in the FBI for convicted felons (i.e.,
CODIS
-
CO
mbined
D
NA
I
ndex
S
ystem, involves 13 STR
loci) and the Dept. of Defense for armed service
personnel and the Virginia saliva and blood bank of
convicted felons


A national DNA database has been suggested. What
do you think?


Could current or potential employers or insurance
companies base decisions they make on this kind of
data?

Fig. 9.18 Random Amplified Polymorphic DNA (RAPD)


Use of arbitrary oligonucleotide primers,
usually 9
-
10 nucleotides long, in a PCR of total
DNA to distinguish plant cultivars, animal
varieties, and microbe isolates


A PCR product will be produced whenever two
of the oligonucleotide primers face one
another and are 100
-
3,000 bp apart

Chromosomal DNA

Region of amplified DNA

Fig. 9.20 Real Time PCR


A way to quantitate
DNA in a PCR


Involves the use of
SYBR green dye


SYBR green only
binds to and
fluoresces with
dsDNA

Fig. 9.16 Bacterial biosensors


One example involves using
Pseudomonas
fluorescens

(genetically engineered for
bioluminescence) to monitor pollutants


If pollutants are present in a sample, then cell
death occurs and “the light goes out”

lux

genes in the

chromosomal DNA

Fig. 9.5 Bacterial biosensors (another example)


Green fluorescent protein (GFP) can be used a
reporter gene under the control of some inducible
promoter (e.g., one that responds to some
environmental signal such as a toxin)


If the signal is present GFP will be produced

Molecular Diagnosis of Genetic Disease


Cystic fibrosis


Sickle
-
cell anemia


(see Fig. 9.28)