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 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)

primers.exponentially increase of the DNAmolecules .PCR • Pre-requisite: -a sequence of nucleotides must be known on both the strands . polymerase and dNTP • binding of primers to ssDNA (annealing) • synthesis of DNA from the primers • incubate a certain time 3) Repeat the process:  .primers (15-25 oligonucleotides) can be made • The process: 1) Heat denature the DNA molecule: 2) Cool and add:  ssDNA .

. cont.PCR.

PCR. cont. .

PCR. . cont.

contamination by ’foreign’ DNA-molecules . cont. 2) The product increases exponentially.the template linearly 3) Problems: . • Note! 1) Taq-polymerase (Thermus aquaticus) is thermo stabile  the reaction can be run at 70-72C.PCR.mistakes in DNA synthesis  use a polymerase with proof-reading .use separate pipettes for PCR .

blood .Some application of PCR 1) Medicine: -diagnose of pathogenic micro organisms .fatherhood If positive PCR Found guilty! .saliva .trace viruses .trace chromosomal aberrations 2) Juridical/forensic medicine: .diagnose of early stages of a bacterial infection .

low amount of DNA  high amount 4) Microbial ecology . cont.Some application of PCR. 3) Molecular biology: • DNA sequencing possible after PCR • Determine chromosomal aberrations • in vitro mutagenesis • construction of vectors ( + restriction sites) .

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

the choice of starting material important .selection of environment (temp.use laser tweezers Note! Important to confirm the purity! .selection of growth medium (amount and chemical form of the nutrients) .) • Isolation .< 1% of natural populations .Bio-diversity Cultivable micro-organisms: • Enrichment .most-probable-number (MNP) ..single-cell-colonies on plates .casting in melted agar (tubes) . pH etc.uses selection and counter selection .

cont.fluorescence .binds to nucleic acids .o.specific groups of m.specific metabolic processes Staining techniques: • DAPI (4’. Non-cultivable micro-organisms: • Quantifying: .Bio-diversity.o.total amount of m.6-diamido-2-phenylindole) .all types of cells (alive and dead) . . .total amount of cells .

• Live/Dead BacLightTM: (contains propidium iodide) • Fluorescent antibodies: .identifying specific m.Bio-diversity.g.o. pathogenic m. cont.red cells  dead cells . clinically.  Molecular methods needed! .green cells  cells alive .e.o. Note! All staining techniques use microscopy  no information about the genetically variation in the population. .

group specific sequences in 16S rRNA as probes (species.different fluorescent dyes attached to the probe .Bio-diversity.hybridization direct to the ribosomes  The whole cell appear fluorescent .the cells are fixated and made permeable to the probe/s . cont. Molecular methods: FISH (fluorescent in situ hybridization) • Species composition of a sample: Use of: .domains) . ….

• Identification of specific genes: Use of: .Bio-diversity.treat the cells as previously for FISH  The gene is present in the population if positive staining! . cont.a fluorescent probe against part of a gene .

cont.Probe 1 against a specific mRNA molecule .FISH .Bio-diversity.Binding + reverse transcription  complementary DNA-strand produced . • Identification of expressed genes: Use of: -ISRT (in situ reverse transcriptase + FISH .DNA synthesis with PCR .a fluorescent dye is added to get probe 2 .

amplify by using PCR .resolving genes of the same size but differing in sequences . cont.group specific primers (16S rRNA genes) How to separate? Use: . • Phylogeny studies: .Bio-diversity.extraction of total DNA in the sample .DGGE (denaturing gradient gel electrophoresis) .

.urea/form amid mixture  ds-DNA  ssDNA at a special conc.heat or . cont.Bio-diversity. Based on: • a denaturing substance/ agent .  Each band can be isolated and sequenced! .

and dTTP • Tube 1: + ddATP Tube 2: + ddCTP Tube 3: + ddGTP Tube 4: + ddTTP . dGTP.DNA sequencing Two methods have been developed: 1) 2) Maxim and Gilbert method Sanger dideoxy method -4 reaction mixtures (tubes) are used . dCTP.nowadays only one tube! 20 Sanger: • ssDNA(/RNA) • primers • DNA-polymerase • a mixture of dATP.

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