Download PCR-technique Applications

PCR-technique
Applications
by
E. Börje Lindström
This learning object has been funded by the European Commissions FP6 BioMinE project
DNA synthesis (replication)
General aspects (in vivo):
• semi conservative
- 1 old + 1 new strand
• template
- the strand that is copied
• replication
-Synthesis in direction 5’3’
- the new strand has a free OH-group
• DNA
polymerase III
- the enzyme needs a primer with a free
OH-group to start
- the primer is RNA (in vivo) or DNA (in
vitro)
PCR
• Pre-requisite:
-a sequence of nucleotides must be
known on both the strands
- primers (15-25 oligonucleotides) can
be made
• The process:
1) Heat denature the DNA molecule:
2) Cool and add:
 ssDNA
- primers, polymerase and dNTP
• binding of primers to ssDNA (annealing)
• synthesis of DNA from the primers
• incubate a certain time
3) Repeat the process:

- exponentially increase of the DNAmolecules
PCR, cont.
PCR, cont.
PCR, cont.
PCR, cont.
• Note!
1) Taq-polymerase (Thermus aquaticus) is thermo stabile 
the reaction can be run at 70-72C.
2) The product increases exponentially- the template linearly
3) Problems:
- contamination by ’foreign’ DNA-molecules
- mistakes in DNA synthesis  use a polymerase with
proof-reading
- use separate pipettes for PCR
Some application of PCR
1) Medicine:
-diagnose of pathogenic micro organisms
- diagnose of early stages of a bacterial
infection
- trace viruses
- trace chromosomal aberrations
2) Juridical/forensic
medicine:
- blood
- saliva
- fatherhood
If positive PCR
Found guilty!
Some application of PCR, cont.
3) Molecular biology:
• DNA sequencing possible after PCR
• Determine chromosomal aberrations
• in vitro mutagenesis
• construction of vectors ( + restriction sites)
4) Microbial ecology
- low amount of DNA  high amount
Microbial ecology
What is studied?
• Bio-diversity
- species variation
• Microbial activity
- nitrogen fixation
- sulphate reduction etc.
Bio-diversity
Cultivable micro-organisms:
• Enrichment
- < 1% of natural populations
- uses selection and counter selection
- the choice of starting material important
- selection of growth medium (amount and
chemical form of the nutrients)
- selection of environment (temp., pH etc.)
• Isolation
- single-cell-colonies on plates
- casting in melted agar (tubes)
- most-probable-number (MNP)
- use laser tweezers
Note! Important to confirm the purity!
Bio-diversity, cont.
Non-cultivable micro-organisms:
• Quantifying:
- total amount of m.o.
- specific groups of m.o.
- specific metabolic processes
Staining techniques:
• DAPI (4’,6-diamido-2-phenylindole)
- fluorescence
- binds to nucleic acids
- all types of cells (alive and dead)
- total amount of cells
Bio-diversity, cont.
• Live/Dead BacLightTM:
- green cells  cells alive
(contains propidium iodide)
- red cells  dead cells
• Fluorescent antibodies:
- identifying specific m.o.
- e.g. clinically, pathogenic
m.o.
Note! All staining techniques use microscopy  no
information about the genetically variation in the population.

Molecular methods needed!
Bio-diversity, cont.
Molecular methods:
FISH (fluorescent in situ hybridization)
• Species composition of a sample:
Use of:
- group specific sequences in 16S rRNA as probes (species, ….domains)
- different fluorescent dyes attached to the probe
- the cells are fixated and made permeable to the probe/s
- hybridization direct to the ribosomes

The whole cell appear fluorescent
Bio-diversity, cont.
• Identification of specific genes:
Use of:
- a fluorescent probe against part of a gene
- treat the cells as previously for FISH

The gene is present in the
population if positive staining!
Bio-diversity, cont.
• Identification of expressed genes:
Use of:
-ISRT (in situ reverse transcriptase + FISH
- Probe 1 against a specific mRNA molecule
- Binding + reverse transcription  complementary DNA-strand produced
- DNA synthesis with PCR
- a fluorescent dye is added to get probe 2
- FISH
Bio-diversity, cont.
• Phylogeny studies:
- extraction of total DNA in the sample
- amplify by using PCR
- group specific primers (16S rRNA genes)
How to separate?
Use:
- DGGE (denaturing gradient gel electrophoresis)
- resolving genes of the same size but differing in sequences
Bio-diversity, cont.
Based on:
• a denaturing substance/
agent
- heat or
- urea/form amid mixture

ds-DNA  ssDNA at a special conc..

Each band can be isolated and sequenced!
DNA sequencing
Two methods have been developed:
1)
Maxim and Gilbert method
2)
Sanger dideoxy method
20 Sanger:
• ssDNA(/RNA)
-4 reaction mixtures (tubes) are used
- nowadays only one tube!
• primers
• DNA-polymerase
• a mixture of dATP, dCTP, dGTP, and dTTP
• Tube 1: + ddATP
Tube 2: + ddCTP
Tube 3: + ddGTP
Tube 4: + ddTTP
DNA sequencing, cont.

Electrophoresis

Analysis
Tube
1(A)
Tube
2 ( C)
Tube
3(G)
Tube
4(T)
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Read from bottom:
C, A, T, G, C, C, A
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