PCR-techniqueApplications

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: ssDNA

2) Cool and add: - 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 DNA-molecules

PCR, cont.

PCR, cont.

PCR, cont.

PCR, cont.• Note!

2) The product increases exponentially- the template linearly

1) Taq-polymerase (Thermus aquaticus) is thermo stabile the reaction can be run at 70-72C.

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 - low amount of DNA high amount

• Determine chromosomal aberrations

• in vitro mutagenesis

• construction of vectors ( + restriction sites)

4) Microbial ecology

Microbial ecologyWhat is studied?

• Bio-diversity - species variation

• Microbial activity - nitrogen fixation

- sulphate reduction etc.

Bio-diversityCultivable micro-organisms: - < 1% of natural populations

• Enrichment - 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:

(contains propidium iodide)

- green cells cells alive

- 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 sequencingTwo methods have been developed:

1) Maxim and Gilbert method

2) Sanger dideoxy method

20 Sanger: -4 reaction mixtures (tubes) are used

- nowadays only one tube!• ssDNA(/RNA)

• 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