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 t e 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 r o p hy l a x i s , a c c u ra t e d i a g n o s i s
a n d e f fe c t i v e t re a t m e n t
 p e r s o n a l i s e d m e d i c i n e - t h e ra p y t a 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
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., Legionella , 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
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 1010 (100mL to 10 fL)
 quantitative subfemtomolar range sensitivity
Immunological methods
 western blotting
 SDS-Page - separates components according to molecular weight
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 Chlamydomonas
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)
Acceptor dye
(Quencher)
Taq
Light Emission
Light
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 a l f e m a l e n o r m a l m a l e
X
Y
1 6
1 3
1 4
n o r m a l 1 3 , 1 4 m o n o s o 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
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 d angerous levels of pathogen s
 EXAMPLE: prion detection
detection limit 100 ng /L
10-fold lower than classical ELISA
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
Nucleic acid diagnostic systems
 polymerase chain reaction (PCR)
 Padlock probe (qPCR/PLP)
o target -complementary sequences
at 5′ and 3′ ends (T1, T2)
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 r o 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 r o p l e t d i g i t a l P C R
 droplets 1 nano liter (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 femto liter (10-15 liters )
 subfemtomolar range sensitivity
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)
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?