PCR-technique Applications

DNA synthesis (replication)

General aspects (in vivo):
semi conservative template replication - 1 old + 1 new strand - the strand that is copied -Synthesis in direction 53 - 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) - low amount of DNA high amount

4) Microbial ecology

Microbial ecology
What is studied?
Bio-diversity Microbial activity - species variation - 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: (contains propidium iodide) Fluorescent antibodies: - green cells cells alive - red cells dead cells - 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) 2) Maxim and Gilbert method Sanger dideoxy method -4 reaction mixtures (tubes) are used - nowadays only one tube!

20 Sanger:
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) ----------------------------------Read from bottom: C, A, T, G, C, C, A Tube 2 ( C) Tube 3(G) Tube 4(T)