Slide 1

TECHNIQUES & TOOLS FOR STUDYING DNA Genomes are very large - so need methods to obtain small (relatively speaking) sections of DNA in abundant & pure form for molecular analysis 1. Restriction enzyme cleavage & agarose gel electrophoresis 4. Molecular cloning 3. PCR (polymerase chain reaction) 2. Southern hybridization Slide 2 1. Restriction enzymes Fig.2.10 blunt endssticky ends 1. DNA endonucleases cut double-stranded DNA at specific recognition sites (often palindromic) - cleave DNA into specific, small fragments 2. Recognition sequences often 4 or 6 bp, but also rare cutters (eg. NotI 5 GCGGCCGC 3 ) can be useful for generating very large fragments in genomic mapping BamHI: staggered cut with 5 overhang Sites are often shown as one strand, but implicit that double-stranded Slide 3 -compatible ends useful for cloning (eg. partial Sau3A genomic digest ligated into BamHI site in vector) 4. Isoschizomers restriction enzymes with identical recognition sequences but may have different response to methylation state MspI cleaves 5 CCGG 3 regardless of methylation state HpaII does not cleave 5 CCGG 3 if 2d C is methylated 3. Two different restriction enzymes may generate same sticky ends Slide 4 - used assay called HpaII tiny fragment Enrichment by Ligationmediated PCR Genome-wide DNA methylation analysis reveals novel targets for drug development in mantle cell lymphoma Example using isoschizomers to assess DNA methylation state of genes in cancer patients Leshchenko et al. Blood 116:1025, 2010 - found significant aberrancy in promoter methylation patterns compared with normal NBCs log(HpaII/MspI) ratios NBC: nave B cells (ie. from healthy people) Slide 5 Agarose gel electrophoresis Fig.T2.2 - to separate DNA fragments by size Fig.T2.1 Small fragments migrate more rapidly than large ones Slide 6 Pulsed field electrophoresis for separation of large DNA molecules Fig.3.30 For example: - restriction fragments generated by rare cutters -megaplasmids -whole chromosomes (eg. yeast) Slide 7 10 kb - 5 kb - 2 kb - 1 kb - 0.5 kb - 20 kb - BS 1 2 3 Lane 1 = uncut DNA Lane 2 = 6 bp cutter Lane 3 = 4 bp cutter so continuum of signals in lane 50 kb - Enzyme with 6 bp recognition site expected to cut a DNA molecule (of 50% GC content) on average once every 4 6 bp (ie. 4096 bp) (see p.86) But if DNA is very complex, number of fragments (of various sizes) generated is too large to see discrete bands after electrophoresis... Why are different profiles expected for genomic DNA cleaved with BamHI (6 bp cutter) vs. Sau3A (4 bp cutter)? Distance (on average) expected between restriction sites depends on probability of occurrence of that sequence Slide 8 2. Southern hybridization 1.After electrophoresis, denature DNA and transfer it from gel to membrane (eg. by capillary action or electroblotting) - to detect specific restriction fragment containing sequence (eg.gene) of interest (vs. all other fragments) Fig.2.11A so that DNA fragments remain in same relative positions Slide 9 2. Hybridize blot with probe (DNA, oligomer, cDNA, or RNA which is tagged either radioactively or non-radiolabelled) and detect specific hybrid by autoradiography Fig.2.11B Stability of hybrid depends on: - length of hybrid, (eg for oligomer probes), GC content - hybridization conditions (such as temperature, ionic strength) - probe will anneal with single-stranded DNA on blot, if sequences are complementary Slide 10 Slide 11 Some applications of Southern blot analysis - to identify restriction fragment carrying sequence (eg gene) of interest - to identify gene copy number (eg. multi-gene families) Aside: Northern hybridization RNA is electrophoresed, blotted to membrane and hybridized with probe (eg gene of interest) - to determine if gene is active, size of mRNA & its abundance (Fig.5.11) (to be discussed in Topic 6) Slide 12 3. PCR - polymerase chain reaction - rapid amplification of DNA region of interest by enzymatic reaction in test tube 1. Denaturation of duplex DNA 2. Annealing of 2 different primers (synthetic oligomers, usually 15-25 nt ) 3. Extension of complementary strands - cycle repeated 25-30 times - flank region of interest, - in opposite orientation Fig.2.28 so anneal to opposite strands of DNA - to obtain one specific DNA region in large copy number "Scientists for Better PCR" a Bio-Rad Music Video for the all new 1000-Series Thermal Cyclers http://www.youtube.com/watch?v=x5yPkxCLads Video at http://www.maxanim.com/genetics/PCR/pcr.swf First cycle Slide 13 Subsequent PCR cycles - discrete PCR product generated - sequences at ends of amplicon correspond to the 2 primers used - its length corresponds to distance between primers (including the primers) Fig.2.29 Slide 14 5 3 5 GATTCC... GCGTAT... CTAAGGCGCATA... How many times would a particular 20mer sequence be expected to be present in the human genome, by chance? Designing PCR primers: Why choose ~ 20mers? Why choose ~ 50% GC? - to reduce chance of non-specific annealing at other genomic sites - for specificity 5 3 Typically use 20-25 nt oligomers, but for simplicity (as on a test) 6mers are shown here (and avoid homopolymeric stretches) Tip: see Question 2.5 in text (p.61) Slide 15 How to double-check that PCR product (amplicon) is correct one? - Southern hybridization - nested PCR - restriction analysis 1. Is it the right size? - agarose gel electrophoresis (with size markers) 2. Does it contain the right sequence (eg gene X)? Fig. 2.30 - using gene X (eg. clone) as probe - are expected restriction sites present? - design internal primers to use in 2d PCR experiment with 1 st PCR product as template DNA New primer Well Slide 16 RT-PCR 5 3 53 5 3 5 3 5 Gene-specific oligomer or oligo dT - (need sequence data to design primers for RT-PCR) - then sequence RT- PCR product directly (or after cloning) 5 3 5 3 5 3 (see p.142, Chapter 5) Slide 17 http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechQPCR.shtml Real-time quantitative RT-PCR eg. SYBR green, TaqMan RFU = relative fluorescence units NTC = no template control Rn : increment of fluorescent signal at each time point - detection and measurement of products generated during each cycle of PCR by using a reporter fluorescent probe NCBI Technologies website CT : PCR cycle number where reporter fluorescence is greater than threshold - to measure relative or absolute amount of mRNA present in different tissue types/developmental stages/environmental conditions Slide 18 Some applications of PCR: - forensic work - paleobiology (ancient DNA) - genomic analysis - RNA studies (RT-PCR) Powers & pitfalls of PCR - rapid method to generate large amounts of specific segment of DNA (product usually < 10 kb in length) - need prior sequence info to design primers but can lead to contamination problems - need very small amount of template DNA Slide 19 Fig. 2.15 4. CLONING - DNA fragments ligated into vector then introduced into bacterial (or yeast) cell to generate clone library by transformation - to obtain one specific DNA region in large copy number - by using host cell (eg. E.coli) to amplify DNA of interest = collection of clones whose inserts cover the entire genome Aside: cDNA library - mRNAs reverse-transcribed into cDNAs and cloned (Fig. 5.32) Slide 20 Examples of cloning vectors used to generate clone library (or bank) 1.Plasmid - to clone < 10 kb fragments - origin of replication, selectable markers eg. antibiotic resistance in bacteria: ampicillin, tetracycline Table 2.4 or nutrient requirement in yeast: URA3, TRP1 Fig.2.18 Insert disrupts lacZ gene, so Xgal on plate not converted to blue colour & colonies are white lacZ = marker for rapid screening of recombinants Slide 21 2. Phage lambda - to clone 15-20 kb DNA fragments Mid-region of DNA molecule can be removed and replaced with similar-sized insert DNA of interest, then packaged in phage particle 3. Cosmid - -plasmid hybrid, cos site to package DNA in phage particle - to clone ~40-45 kb fragments 4. BAC - bacterial artificial chromosome (~8 kb) with F (fertility) plasmid origin of replication - to clone ~ 300 kb fragments (Aside: also vectors for cloning cDNAs of 1-5 kb) - most commonly used vector for cloning large DNA fragments Slide 22 5. YAC - yeast artificial chromosome - to clone ~ 1 Mbp fragments - but sometimes DNA rearrangements & instability of inserts Fig.2.25 Selectable markers (TRP1 & URAS3) - yeast host strain requires tryptophan & uracil in medium to grow, but transformants (which possess TRP1 & URA3 genes on YAC) can grow in medium lacking them Slide 23 How many clones needed in library to cover a complete genome? Depends on: genome sizeand insert size in vector N = ln (1 P) ) ln (1 insert length/ genome length) Number of clones N that must be screened to isolate a given sequence with a probability of P: Rule-of-thumb: For 99% probability of success, the total # bp present in clones screened must be about 5 x greater than total genome size For E.coli (genome size ~ 4.6 Mbp), how many clones needed if average insert size = 10 kb? Table 2.4 Slide 24 Strategies to generate overlapping clones? A B A A B A B B A B A A B A B DNA cleaved with A DNA cleaved with B etc. prepare library A B B B 1. Use two clone banks with restriction fragments derived from different restriction enzymes (or from incomplete digestion with one restriction enzyme) - use in assembling genomic maps Then can look for overlapping clone in B library... Can use clone from B library as probe to find clone in A library that contains part of the same sequence plus neighbouring sequences chromosome walking Slide 25 Cold Spring Harbor Protocols 2010 Nebulizer for random shearing of DNA 2. Random fragmentation of DNA (eg sonication or nebulization ) then blunt-end ligation into vector (or ligation into vector after linkers containing restriction sites added) 1 kb Recover DNA of desired size (eg. 1 kb) by gel electrophoresis (or repeated nebulization) & prepare clone library... having random overlapping segments of genome Aside: this method was used to obtain the first complete bacterial genome sequence (Haemophilus influenza) (Topic 5 & Fig.4.10)