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Bi7430 Molecular Biotechnology
10. Molecular Biotechnology
in Medicine
Outline
 definition of red biotechnology
 areas of red biotech applications
 molecular diagnostics
 immunological diagnostic methods
 nucleic acid diagnostic systems
 digital diagnostics
 personalized medicine
Red (medical) biotechnology
 b i o te c h n o l o g y t h a t d e a l s s p e c i f i c a l l y w i t h h u m a n h e a l t h
c a re a n d m e t h o d s o f t re a t m e n t
 a i m a t p ro p h y l a x i s , a c c u ra te d i a g n o s i s
a n d e f f e c t i v e t re a t m e n t
 p e rs o n a l i s e d m e d i c i n e - t h e ra p y ta i l o re d b a s e d o n p a t i e n t
p ro f i l e ra t h e r t h a n t h e “o n e s i ze f i t s a l l ” a p p ro a c h
 p ro m i s i n g a re a s o f re d b i o te c h a p p l i c a t i o n s :
 m o l e c u l a r d i a g n o s t i c s a n d g e n e t i c t e s t i n g
 v a c c i n e s , p r o t e i n a n d n u c l e i c a c i d t h e r a p e u t i c s
 t i s s u e e n g i n e e r i n g a n d r e g e n e r a t i v e m e d i c i n e
 g e n e t h e r a p y a n d t h e r a p e u t i c a l c l o n i n g
 d r u g d e l i v e r y a n d n a n o m e d i c i n e s
Clinical diagnostics
 success of modern medicine depends on specific detection of
 viruses
 bacteria
 fungi
 proteins
 nucleic acids
 medical laboratory methods contribute to 80% of diagnosis
 good detection method should have three characteristics
 sensitivity - ability to detect small amounts of target molecule
 specificity - positive result for the target molecule only
 simplicity - ability to run efficiently, inexpensively on a routine basis
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Clinical diagnostics
 classical methods
 cultivation, microscopic analysis, biochemical assays
 POSITIVES: simple, direct detection
 NEGATIVES: slow, laborius, low sensitivity, high skill level
requirement, dengerous during cultivation infectious organisms
 molecular diagnostics (past 20 years)
 immunological and nucleic acid diagnostic systems
 POSITIVES: fast, simple, high sensitivity, automatable, safe
 NEGATIVES: not always specific, possible false possitive
or negative results
Immunological methods
 sensitive, specific and simple
 based on antigen-antibody interactions
 protein >> sugar > nucleic acid
 wide range of applications in monitoring:
 hormones, vitamins, metabolites, diagnostic markers
(e.g., insulin, testosterone, prostaglandins , corticoids)
 drugs
(e.g., barbiturates, morphine, digoxin)
 infections
(e.g., Leg ion ella , HIV, hepatitis A, B)
 cancer
(e.g., alpha-fetoprotein, carcino-embryonic antigen)
Immunological methods
 agglutination
 blood typing test
 ABO blood-group antigens
(differences in the sugars on glyco-proteins)
Immunological methods
 agglutination
 pregnancy test
 inhibition in presence of human chorionic gonadotropin,
hCG, glycoprotein hormone produced in pregnancy
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Immunological methods
 immuno-chromatographic assays
 simple devices to detect presence of analyte in sample
 no need for specialized equipment or sample treatment
 coloured particle - latex (blue) or nanosized gold (red)
 sandwich double antibody reaction scheme (e.g. HIV, hCG)
 competitive reaction scheme (small antigens)
Immunological methods
 enzyme-linked immunosorbent assay (ELISA)
 enzyme based detection (e.g., HRP, β-galactosidase, phosphatase)
 florescence or colorimetric based detection
Immunological methods
 digital immunoassay (single molecule ELISA)
 detection volume decreased by a factor of 10 10 (100mL to 10 fL)
 quantitative subfemtomolar range sensitivity
Immunological methods
 western blotting
 SDS-Page - separates components according to molecular weight
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Immunological methods
 western blotting
 SDS-Page - separates components according to molecular weight
 Blot: proteins in gel transferred to nitrocellulose or nylon
Immunological methods
 western blotting
 SDS-Page - separates components according to molecular weight
 Blot: proteins in gel transferred to nitrocellulose or nylon
 Immunoreaction: after blocking (BSA) probed with primary
and secondary antibody
 Detection: radioactivelabelling, colorimetry, florescence,
(chemi)luminescence
Immunological methods
 immunoprecipitation
 collected by magnetic beads coupled to a secondary antibody
Immunological methods
 immunofluorescence (microscopy methods)
 fluorescence labelled antibody (e.g., fluorescein, rhodamine)
lacrimal gland myoepithel virus infected cells Ch la myd omon a s
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Nucleic acid diagnostic systems
 most common object for testing is DNA, in some cases RNA
 areas of medical applications:
 prenatal diagnostics: non-invasive detection of fetal diseases
(e.g., Down syndrome, cystic fibrosis)
 genetic testing: high throughput testing for genetic disorders
(e.g., SNPs markers, insertions, deletions)
 infectious diseases: pathogen identification and drug resistance
(e.g. HIV, HBV, HCV)
 oncology: early diagnosis of cancer
(e.g., circulating tumor DNA, ratinoblastoma gene)
 transplantation medicine: non-invasive detection of organ rejection
(e.g., urine testing for kidney rejection, human leukocyte antigen)
 pharmacogenomics: influence of genetic variation on drug response
 DNA typing: fingerprint of genotypic traits
(paternity, crime suspects, ancestry)
Nucleic acid diagnostic systems
 DNA hybridization
 probe which anneals to the target nucleic acid
 bacterial and viral pathogens contain specific gene(s)
 genetic diseases caused by mutation or absence of particular gene(s)
Nucleic acid diagnostic systems
 DNA hybridization
 conventional method
1. attachement of target DNA to solid matrix
2. denaturation of both probe and target
3. annealing probe to target DNA
4. washing and detection
(e.g. autoradiography, chemoluminiscence , fluorescence )
Nucleic acid diagnostic systems
 DNA hybridization
 conventional method
 TaqMan Probes - hydrolysis by Taq polymerase
Taq
Donor dye (Reporter)
Acceptordye
(Quencher)
Taq
Light Emission
Light
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Nucleic acid diagnostic systems
 DNA hybridization
 conventional method
 TaqMan Probes - hydrolysis by Taq polymerase
 molecular beacons - hairpin DNA with internally quenched fluorophore
Nucleic acid diagnostic systems
 DNA hybridization
 EXAMPLE: detection of parasite Plasmodium falciparum
 microscopic observations of blood smears is labour intensive
 ELISA does not differentiate between past and present infection
 DNA diagnostic system measure only current infection
Other examples: Salmonella typhi (food poisoning)
Escherichia coli (gastroenteritis)
Nucleic acid diagnostic systems
 DNA hybridization
 fluorescence in situ hybridisation (FISH)
 new technique for karyotyping
 chromosome abnormalities (segmental deletions and translocations )
 aneuploidy (abnormal number of chromosomes)
n o r m al fe m al e n o r m al m al e
X
Y
1 6
1 3
1 4
n o r m al 1 3 , 1 4 m o n o so my 1 4
Nucleic acid diagnostic systems
 DNA hybridization
 DNA microarray (DNA chip)
 104 to 106 probes (reporters)
 spot - picomole (10-12 M) of oligo
 probe-target hybridization
 labelling by chemiluminescence ,
fluorophore or silver
 bioinformatics data processing
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Nucleic acid diagnostic systems
 polymerase chain reaction (PCR)
 amplify single or few copies of DNA to millions of copies
 the presence of the appropriate amplified size fragment
(product) confirms the presence of the target
 specific primers are available for detection of
bacteria (E. coli, M. tuberculosis), viruses (HIV), fungi
 early diagnosis of malignant diseases ( leukemia)
Nucleic acid diagnostic systems
 polymerase chain reaction (PCR)
 real-time PCR (qPCR)
o non-specific fluorescent dyes that intercalate with dsDNA
o sequence-specific DNA probes, oligonucleotides labeled with
fluorescent reporter
Nucleic acid diagnostic systems
 polymerase chain reaction (PCR)
 reverse transcription PCR (RT-PCR)
 real-time reverse-transcription PCR (qRT-PCR)
Nucleic acid diagnostic systems
 polymerase chain reaction (PCR)
 immunoquantitative real-time PCR (iqPCR)
o combines specificity of antibodies and sensitivity of PCR
o overcome insufficient sensitivity of available immunological methods
o sensitive for very low but still dangerous levels of pathogen s
 EXAMPLE: prion detection
detection limit 100 ng /L
10-fold lower than classical ELIS A
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Nucleic acid diagnostic systems
 polymerase chain reaction (PCR)
 random amplified polymorphic DNA (RAPD)
o „random“ primers used to produce DNA fingerprint
o primers anneal in many places on template DNA
and produce variety of sizes of amplified products
DNA fingerprinting
Nucleic acid diagnostic systems
 polymerase chain reaction (PCR)
 restriction fragment length polymorphism (RFLP)
o many diseases caused by single nucleotide change
o method dependent on mutation within recognition site
of restriction enzyme
 EXAMPLE: diagnostics of sickle cell anemia
o anemia and damage to heart, lung,
brain, joints and other organs
o single nucleotide change in 6th aa
of beta-chain of hemoglobin (E6V)
o normal DNA sequence CCT GAGG (A)
o mutant DNA sequence CCT GTGG (S )
o homozygous state SS red blood cells
irregularly shape d
NORMAL SICKLE-CELL
Nucleic acid diagnostic systems
 polymerase chain reaction (PCR)
 oligonucleotide ligation assay (PCR/OLA )
Nucleic acid diagnostic systems
 polymerase chain reaction (PCR)
 Padlock probe (PCR/PLP)
o target-complementary sequences
at 5′ and 3′ ends
o ligate only if perfect match
o only ligated forms attach to target
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Nucleic acid diagnostic systems
 polymerase chain reaction (PCR)
 Padlock probe (qPCR/PLP)
o target-complementary sequences
at 5′ and 3′ ends (T 1, T 2)
o universal primer sites (P1, P2 )
o reporter sequence (Zip)
o only ligated forms attach to target
Szemes et al. 2005. Nucl. Acids Res. 33: e70
Nucleic acid diagnostic systems
 DNA sequencing
 most direct method
 become cheap and fast, pushes other methods backward
 genes, genetic regions (i.e. gene clusters or operons),
full chromosomes or entire genomes
 EXAMPLE: Diagnostic for Duchenne muscular dystrophy (DMD)
 mutated distrophin (”implosion” of muscle cells )
 X-linked recesive, carrier mother
 dystrophin gene large (2,4 Mb )
 first mutation carrier often mosaic
(blood may be not a mutation carrier)
Nucleic acid diagnostic systems
 DNA sequencing
 2003 Human Genom Project (13 years)
 2008 Jamese Watson genom (6 month)
 2015 genome sequencing in (10 hours)
Digital diagnostics
 s i n g l e m o l e c u l e p y ro s e q u e n c i n g
 droplets 1 picoliter (10-12 liters)
 1 mil. reads/run, 1-10 USD/Mbase
 d ro p l e t d i g i t a l P C R
 droplets 1 nanoliter (10-9 liters)
 20 thousand reads/run
 s i n g l e m o l e c u l e E L I S A
 volume 10 femtoliter (10-15 liters)
 subfemtomolar range sensitivity
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Personalized medicine
 medical practice and products tailored to individual patient
 genetic, molecular or cellular diagnistics
 genetic information has major role in personalized medicine
(e.g. pharmacogenomics)
 miniaturization / simple handheld devices
 medical diagnostics from hospital/clinics to office/home
Personalized medicine
 pharmacogenomics
 designing the most effective drug therapy
based on specific genetic profile of patient
 different drug effects - genetic polymorphisms
Personalized medicine
 pharmacogenomics
 personalized oncology
 analyse tumor
 design specific treatment
Personalized medicine
 pharmacogenomics
 personalized oncology
 pre-implantation genetic diagnosis (PGD)
 7000 genetic deseases 4000 known (Mendelianian heretige)
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Personalized medicine
 pharmacogenomics
 personalized oncology
 pre-implantation genetic diagnosis (PGD)
 gene editting (gene therapy)
Personalized medicine
 pharmacogenomics
 personalized oncology
 pre-implantation genetic diagnosis (PGD)
 gene editting (gene therapy)
 WHAT NEXT?