1. PCR-technique Applications by E. Börje Lindström This learning object has been funded by the European Commissions FP6 BioMinE project

2. 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)

3. 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

4. PCR, cont.

7. 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

8. 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!

9. 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

10. Microbial ecology What is studied? Bio-diversity- species variation Microbial activity- nitrogen fixation - sulphate reduction etc.

11. Bio-diversity Cultivable 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!

12. 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

13. Bio-diversity, cont. Live/Dead BacLight TM : (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!

14. 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

15. 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!

16. 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

17. 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

18. 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!

19. DNA sequencing Two methods have been developed: 1)Maxim and Gilbert method 2) Sanger dideoxy method 2 0 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

20. DNA sequencing, cont. Electrophoresis Analysis Tube 1(A) Tube 2 ( C) Tube 3(G) Tube 4(T) ------ ----- ------ Read from bottom: C, A, T, G, C, C, A