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Dr. M. Sasvári : Recombinant DNA Technology, I.
Polymerase Chain Reaction (PCR)
New Tool in Molecular Diagnostic
Http://www.biokemia.sote.hu
Amplification of a desired dsDNA fragment
PCR
Total human genome
2 x 3x109 bp
2 x 23 chromosomes
(2 x ) 102-103 bp PCR product
dsDNA fragment
Specificity of PCR amplification
DNA
DNA
: „oligo” or primer, 16-20 bp synthetic ssDNA
At least two
select the two ends of the PCR product
?
2. cycle
DNA
DNA
PCR amplification is exponential (duplicating in every cycle)
1 dsDNA  106 fragments / 25 cycles (approx.. 3 hours)
The PCR reaction
Metal thermoblock block
(fast changes in temperature)
20-50 ml of reaction mixture
• template DNA
• primers
• dNTP + Mg2+
• Taq DNA polymerize (Thermus aquaticus)
HEAT STABLE
The thermocycle
I. Denaturation (96oC)
dsDNA  ssDNA
T
I.
III.
II.
t
II. Annealing40-60oC)
primers bound
to the complementary sequences
III.Polymerization (72oC)
DNA replication
with the heat stable polymerase
Medical application of PCR
Genetic canceling
Screening for disease causing mutations
Monogenic disorders
lethal/treatable diseases: prenatal diagnosis
Risk factors
Polygenic inheritance
E.g. oncogenes
The DNA sample
invasive DNA sampling:
blood (Guthrie spot)
CVS (chorion villi sample)
non-invasive sampling
Buccal cells
hair, nail
Medical application of PCR
Identification of a foreign genome
Bacterial and viral sequences: not found in the human genome
Detection: specific primers - is there any PCR product?
Examples:
Pneumococcus (tubercolosis) - “sputum”
HIV (?)
Clamidomas
Other applications
DNA “fingerprinting”
Paternity cases
Criminology (hair, blood-spot, etc.)
Immigration (relationship)
Separation and visualization of PCR products
agarose gel
Ethidium bromide
staining (intercalation)
100 V
–
+
Big
UV
Small
ladder
Methods of mutation analysis
PCR-RFLP
e.g.:Sickle cell disease (SSD), hemoglobin b-chain Glu6Val6
1. step: PCR amplification :
A
T
normal gene
T
A
3. step: gel electrophoresis
patient’s number
2.
4.
1.
3.
Mutant gene
2. step: Cleavage with Mst II. restriction endonuclease:
+
Q: Which patient is a carrier?
Who will have SSD?
Detection of a deletion
e.g.: Cystic fibrosis DF 508: 3 bp deletion (Phe minus)
Primer construction :
Gel electrophoresis
1. 2. 3. 4.
CFTR gene
normal product
mutant product
+
Q:If this is a prenatal diagnosis (parents: 1 and 2), which
sample would belong to a healthy embryo (3 or 4)?
Identification of larger deletions
e.g.: DMD (Duchenne muscular dystrophy)
X-linked inheritance ( DMD frequency 1: 3500 men)
The longest known gene (2 million bp)
Very long protein product: Dystrophin (Mw 426 kD)
Deletion sites
A
B
C
1. 2. 3. 4.
Primer construction
Primers are complementary to the deleted sequences
Q: Which of the patients has DMD? Who has probably the most sever symptoms?
Identification of mutations
ASA: Allele Specific Amplification
Principle of ASA: if the 3`end of the primer is not complementary to the template,
no PCR product is formed
Medical importance of prenatal diagnosis
in case of 21-steroid hydroxylase deficiency (CYP21 gene)
CYP 21 mutations: disturbance of steroid metabolism
Underproduction of aldosterone  SALT WASTING
Overproduction of androgenes  VIRILIZATION
Prenatal diagnosis - fetal treatment in females
ASA: Allele Specific Amplification
...
DNAN
...
DNAN
...
DNAMt
...
DNAMt
...
PN: normal primer
3’
PMt: mutant primer
STOP
...
3’
PN: normal primer
...
3’
...
PMt: mutant primer
3’
STOP
Genotyping with ASA
PN
PMt
PN
PMt
PN
PMt
Internal control
Allele specific product
Patient 1
Patient 2
Patient 3
Q: Which of the patients are healthy/carrier/sick (homozygous for the mutation)?
Recombinant DNA technology
Production of human recombinant proteins
for pharmaceutical purposes
Principles of vector construction
Restriction endonuclease(RE)
Foreign DNA
RE
Plazmid
vector
AmpR
Ligation
DNS ligase
recombinant
vector
Expression of a Human gene in Bacteria
Expression vectors
- insert contains only exons (cDNA)
- bacterial promoter
- bacterial ribosome binding sites
Turning on and off the protein production
Bacterial DNA
lac promoter lac operator cDNA of Human protein
Repressor
protein
+IPTG
induction
expression
Insulin: The first licensed recombinant protein
1. Vector construction
lacZ
(b gal)
Artificial “ insulin gene”
AmpR
ori
Insulin
A or B chain
(insert)
Amino acid sequence

base sequence
2. Transformation, selection of AmpR clones, cloning
E. coli
plasmid
AmpR
lac repressor
Bacterial chromosome
3. IPTG added  fusion protein is produced
bgal
4. Lysis, isolation and purification of the protein
5. CN Br treatment: Methionins are broken, insulin released
6. A and B chain fold in vitro
insulin
Bacterial production of Met-less hGH
1. vector construction
hGH
Bacterial
signal sequence
(extracellular)
1. Transformation
selection
growth
2. Secretion of hGH
into the periplasmic space
AmpR
ori
3. Bacterial periplasmic protease:
cuts the signal sequence
4. Met-less rec-hGH, purification
Expression of human gene in mammals
Transient expression
Non-integrating viral vectors
e.g. adenovirus
advantage: no transformation
disadvantage: temporal
Transgenic animals
Microinjection
of the fertilized egg
advantage: stabile integration
random integration
Embrionic Stem cells
(ES cell transformation:
homologue recombination)
‘Knockout’ and ‘knockin’ animals
Transient expression: reporter genes
EuK
cell
Viral-vector-reporter gene construct
nucleus
Expression assay based on the reporter protein
Reporter proteins
• luciferase
ATP  ADP + Pi + light
• b-galactosidase Lactose analogue  blue product
• CAT (chloramphenicol acetyl transferase)
BLUE CELLS
Application of reporter genes
e.g. testing trans -activating proteins (TF)
1
2
3
reporter
1: enhancer
2: promoter
3: gene of TF
vector A
EuK
cell
TF
nucleus
Vector B
Transgenic animals
1982: The giant mouse
Fertilized
oocyte
Suction pipette
vacuum
Microinjection
of hGH cDNA
into the male pronucleus
Preparation of transgenic animals
1. Microinjection (many hundreds of eggs, micromanipulator)
2. Implantation of the egg into a pseudo-pregnant mouse
3. Check the pubs (PCR from the tail)
4. Mate with transgenic animal
5. Inbreeding - Cloning as a new alternative ?
Production of secretory recombinant proteins
Microinjection of mouse with t-PA gene
+ lactalbumin signal sequence and a proper regulatory unit
secretion of t-PA into the milk: 0.1 mg t-PA/ml milk of the mouse
Future: transgenic (cow?), goat, sheep
Genetically modified crops
Molecular Farming
Basic terms (see Lehninger: Recombinant DNA technology)
Vector: • a circular dsDNA molecule
• suitable for inserting a DNA fragment
Maximal size of DNA
• replicates after introducing into a host cell that can be cloned in vectors
Types of vectors:
• plasmid (extrachromosomal DNA)
• phage (bacterial host)
• virus (plant, animal, human hosts)
Genomic library
A series of recombinant phages/cosmids containing
different DNA fragments of the human genome
Cosmid:
• artificial plasmid
plasmid
- phage
cosmid
20 kb
25 kb
45 kb
YAC
1000 kb
(yeast artificial chromosome)
Restriction enzymes
recognition site / fragment size
4 bp
256 bp
6bp
4000 bp
8 bp
64000 bp
• a combination of phage COS sequence + a plasmid
• similar to plasmids: antibiotic resistance gene
replication origo
• can take a large insert (40 kb)
• similar to phage: infects E. coli after packaging in vitro