PCR and recombinant DNA techniques

Transcription

1 PCR and recombinant DNA techniques Cristiano V. Bizarro Joan Camuñas Felix Ritort Group Small Biosystems Lab Universitat de Barcelona

2 Topics to be covered Introduction (DNA isolation, modification enzymes) PCR DNA Electrophoresis Recombinant DNA technology Applications in single-molecule biophysics

3 Zooming in on cellular macromolecules The interior of cells is replete with macromolecules: macromolecular crowding. Complex to investigate the details of the system. Most biochemical procedures require obtaining a large number of cells and then physically disrupt them to isolate its components in pure form. We will review some techniques used to study DNA, RNA and proteins. Gierasch LM et al. Nature Chemical Biology. 2009;5:774-7.

4 DNA structure I Nucleic Acids are created by joining together nucleotides with a phosphodiester linkage: Source: Molecular Biology of the Cell. Alberts et al. Garland Science.

5 DNA structure II Source:Color Atlas Of Biochemistry 2d ed - Jan Koolman, Klaus-Heinrich Rohm

6 DNA isolation techniques -Genomic DNA: -Plasmidic DNA : DNA Lisis: detergents + proteinase K+ enzymes Phenol:Chloroform extraction Ethanol precipitation DNA DNA Aqueous phase Organic phase Small circular DNA molecules found in prokaryotes independent of genomic DNA. Lisis: detergents + NaOH (basic ph) Renaturalization: AcNa (acid ph) 4. RNAse treatement 2. Centrifugation Phenol:Chloroform extraction RNAse treatement Isolated plasmid 6. Ethanol precipitation Isolated DNA Of great interest in molecular biology as they are easy to manipulate, and are mantained and replicated in cells.

7 Recombinant DNA technology Source:Color Atlas Of Biochemistry 2d ed - Jan Koolman, Klaus-Heinrich Rohm

8 Modification enzymes in molecular biology I +

9 Modification enzymes in molecular biology II Major types: - Exonucleases: digest DNA from an end of a strand in the 5 3 direction. Useful to generate blunted-end fragments. -Endonuclases: cut DNA at internal sites. Restriction endonucleases are an important group of endonucleases - Polynucleotide kinase (PNK): Specifically phosphorylate the 5 terminus of a DNA sequence (Alkaline phosphatase catalyzes the reverse reaction).

10 Restriction endonucleases II Type II restriction endonucleases: - Usually recognize 4-8 bp sites on DNA - Most of the sites are palindromic sequences - Some enzymes recognize degenerate sites - Cleavage products contain 5 -PO 4 and 3 - OH termini - Three types of cleavage products: - Blunt-ends - 5 protudent ends - 3 protudent ends

11 Restriction endonucleases I Source:Color Atlas Of Biochemistry 2d ed - Jan Koolman, Klaus-Heinrich Rohm

12 Modification enzymes in molecular biology III Major types: -T4 DNA Polymerase: Catalyzes DNA polymerization in the 5 3 direction using the complementary strand as a template. -(It also has 3 5 exonuclease activity) -DNA ligase: Catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in dsdna.

13 Target DNA genomic DNA A clone from a genomic library containing the sequence of interest A clone from a cdna library Synthetic sequence (normally when the sequence of interest are short: nt)

14 Polymerase Chain Reaction (PCR) Problem: Modern instrumentation cannot easily detect single molecules of DNA, making amplification a prerequisite for further analysis. Solution: PCR doubles the number of DNA fragments at every iteration. Selected DNA fragments can be cloned in a test tube Source:

15 PCR: basic steps Geometric amplification (2 n ) of a specific region of a DNA template delimited by the primer sequences. Primers: Two sets of DNA oligonucleotides (ssdna) that flank the DNA fragment to amplify. Non-specific sequences are amplified linearly (2 n). Source: Molecular Biology of the Cell. Alberts et al. Garland Science. Number of molecules:

16 PCR: basic steps Usually, each cycle of amplification has 3 steps: - denaturation - annealing - elongation (72 ºC) Thermostable DNA polymerases can resist multiple cycles of amplification (eg Taq DNA polymerase, Pfu DNA polymerase)

17 PCR: primer design DNA-dependent DNA polymerases are not able to do de novo synthesis: some molecule should serve as a starting point (primer) to DNA synthesis Primer design: - Length: bases, Tm > 50ºC - 50% GC content - Try to avoid sequences that contain internal complementarity or that have propensity to form dimers - In the first bases, sequence divergence with target DNA are not allowed - Divergent sequences can be added at the 5 terminus

18 Agarose DNA electrophoresis I DNA electrophoresis is an technique used to separate a mixture of DNA molecules by size. -Electrophoresis set-up: sample sample Molecular weight marker DNA (negatively charged) -Fluorescent dye: agarose gel imaging Source:Color Atlas Of Biochemistry 2 nd ed. - Koolman J et al.

19 Agarose DNA electrophoresis II Agarose gel electrophoresis are usually done in an horizontal system; The agarose gel is submersed in a buffer and contain wells to apply the samples;

20 Agarose DNA electrophoresis III Linear, supercoiled and circular relaxed DNA molecules display different electrophoretic mobilities

21 Recombinant DNA technology Source:Color Atlas Of Biochemistry 2d ed - Jan Koolman, Klaus-Heinrich Rohm

22 Cloning vectors Cloning vector: A self-replicating genetic element into which a foreign DNA fragment can be inserted (e.g. plasmids, phages, combinations) Host cells: Bacteria, yeast, mammalian, Plasmids: Plasmid: example - bacterial extrachromosomal elements - Antibiotic resistance - High or low copy number - Multiple cloning site (MCS)

23 Recombinant DNA technology: cloning

24 DNA cloning: basic steps I Example of possible steps to clone a sequence: 1. Design primers containing selected restriction sites at the 5 -termini (must be absent in the target sequence);

25 DNA cloning: basic steps II 2. Isolate genomic DNA; 3. Amplify by PCR the sequence of interest; 4. Visualize the PCR product by agarose gel electrophoresis;

26 DNA cloning: basic steps III 5. Purify the PCR product and digest with restriction endonucleases (EcoRI and HindIII in the example): 6. Digest the cloning vector with the same enzymes: A purification step after agarose gel electrophoresis may be done to get rid off the small fragment

27 DNA cloning: basic steps IV 7. Proceed to the ligation reaction: Add vector and insert containing compatible ends, enzyme reaction buffer, ATP, and T4 DNA ligase; Incubate 16 ºC (usually overnight but can be done in 2 hours). Thermally inactivate DNA ligase (10 min, 70 ºC) 8. Transform the ligation reaction in a suitable bacterial host cell: Different bacterial strains with specific properties; Different protocols to prepare competent cells.

28 DNA cloning: basic steps V 9. Incubate cells in growth medium for 1h at 37ºC and plate onto solid selective media (usually LB plates containing the antibiotic resistance marker present in the cloning vector); 10. Growth selected clones in liquid media (in a small scale 3 ml) and isolate recombinant DNA (miniprep) 11. Confirm that the expected recombinant clone was produced: digestion with restriction endonucleases, PCR, and sequencing

29 Recombinant DNA technology: libraries DNA library: Comprehensive collection of cloned DNA fragments from a cell, tissue or organism. -genomic libraries: The entire genome is cleaved with a restriction endonuclease and fragments are cloned to bacterial cells (shotgun approach). Some clones contain genes and others non coding DNA. cdna libraries: - Contains the set of expressed sequences from a particular source material (cell, tissue, developmental stage, treated/ untreated etc)

30 Molecular synthesis techniques in single-molecule biophysics: examples What can be done with optical tweezers? Molecular constructs for single-molecule experiments: o o Stretching dsdna molecules using optical tweezers Single-molecule analysis of DnaB helicase activity The synthesis of substrates for these experiments requires the use of molecular synthesis techniques of nucleic acids

31 What can be done with op/cal tweezers? Following transla-on by single ribosomes one codon at a -me It is extremely difficult to follow the steps of ribosomes during transla/onal elonga/on using ensemble methods, as it is impossible to synchronize their ac/vity Each step corresponds to the breaking of three hairpin base pairs (2.7nm) Wen JD et al. Nature. 2008;452: Pause lengths depend on the secondary structure of the mrna

32 Stretching dsdna: experimental setup Experiment To study the elastic properties of dsdna using optical tweezers, the molecule must be specifically attached to beads at both ends. Molecular construct to synthesize Tag to bind to bead 1 (400bp) Molecule (24509bp) Tag to bind to bead 2 (400bp)

33 Stretching dsdna: digoxigenin labeling DNA labeling with digoxigenin (only in one end): One bead is revested with anti-digoxygenin antibodies:

34 Stretching dsdna: biotin labeling DNA labeling with biotin (the other end): The other bead is revested with streptavidin:

35 dsdna construct design and synthesis

36 dsdna construct design and synthesis Lambda-DNA: Comercially available DNA from Enterobacteria Phage Lambda (48kbp). cosl and cosr: 12-nt cohesive ends intrinsic to lambda-dna.

37 dsdna design and synthesis: PCRs

38 Incorporation of modified nucleotides Digoxigenin Biotin 1.Marker VII 2.Digoxigenin- 70 µm 3.Digoxigenin - 35 µm 4.Digoxigenin - 17,5 µm 5.Digoxigenin -0 µm 1.Marker VII 2.Biotin- 0 µm 3.Biotin µm 4.Biotin µm 5.Biotin - 87,5 µm 6.Biotin- 43,75 µm 7.Biotin - 0 µm

39 DnaB helicase Helicases are enzymes that move along single strands of DNA and are able to separate the two DNA strands by breaking the H-bonds between them (up to 1000bp/s). Helicases play a role in several cellular processes (e.g. DNA replication, recombination, transcription) DnaB helicase from Aquifex aeolicus VF5 Hexameric replicative helicase

40 DNA helicase substrate Marker VII 2. λdna\sph I 3. SphI fragment

41 Experimental setup