Computational Biology I LSM5191

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1 Computational Biology I LSM5191 Lecture 5 Notes: Genetic manipulation & Molecular Biology techniques

2 Broad Overview of: Enzymatic tools in Molecular Biology Gel electrophoresis Restriction mapping DNA Cloning Polymerase Chain Reaction Constructing DNA libraries Southern Blotting Northern Blotting Sequencing DNA Microarrays

3 Enzymatic tools used in molecular biology Ligases Repairs ss breaks (discontinuity) in ds DNA. Polymerases DNA polymerase I attaches to short ss region (or nick) in a mainly dsdna molecule, its polymerase activity synthesizes new strand on a ssdna template, its nuclease activity degrades the existing (old) strand as it proceeds. Use: e.g. labeling probes by nick translation. Klenow fragment (modified DNA pol I 323a.a region removed) lacks nuclease activity, retains polymerase activity (as in DNA pol I). Use: e.g. labeling probes by end-filling of stagged/sticky ends. Reverse transcriptase RNA-dependent DNA polymerase.

4 Enzymatic tools used in molecular biology DNA modifying enzymes Alkaline phosphatase Removes 5 -terminal phosphate group Polynucleotide kinase Adding phosphate groups on to free 5 -termini. Terminal deoxynucleotidyl transferase Adds one or more deoxynucleotides to the 3 -end

5 Enzymatic tools used in molecular biology Nucleases Exonucleases Exonuclease III: removes nucleotides only from the 3 -end. Bal31: removes nucleotides from both strands Endonucleases S1 nuclease: cleaves only single-stranded (ss) DNA or existing ss nicks, in a mainly dsdna molecule. DNase I: cleaves both ss and double-stranded (ds) DNA. Restriction endonuclease: cleave dsdna at specific/limited no. of sites.

6 Enzymatic tools used in molecular biology Type II Restriction endonucleases: Recognition sequence is usually a palindrome, e.g. EcoRI recognises: 5 -GAATTC-3 3 -CTTAAG-5 Isoschizomers refer to enzymes that recognize the same recognition sequence. Frequency of cutting: 6-bp recognition sequence is likely to occur once every ~4kb (4 6 ), 5-bp recognition sequence is likely to occur once every ~1kb (4 5 ) Sticky ends generated by enzyme: e.g. EcoRI Blunt ends generated by enzyme: e.g. SmaI 5 -N-N-N-C-C-C-G-G-G-N-N-N-3 3 -N-N-N-G-G-G-C-C-C-N-N-N-5 Sticky ends increase the efficiency of ligation. Adapted from Lodish et al., Molec. Cell Biol. WH Freeman.

7 Selected Restriction enzymes and their recognition sites Adapted from Lodish et al., Molec. Cell Biol. WH Freeman.

8 GEL ELECTROPHORESIS Adapted from Brown, 2001, Genomes 2 Ed., Wiley Co. and Lodish et al., Molec. Cell Biol. WH Freeman.

9 GEL ELECTROPHORESIS More accurate graphical estimation 23130bp Rough estimation by eye Est 5000bp Est 3200bp Est 2000bp Size of DNA (kb) Distance migrated (cm) 564 Log 10 nucleotide pairs 0.5% 0.7% 0.9% 1.2% 1.4% Gel concentration Distance migrated (cm)

10 Restriction Mapping

11 DNA CLONING in bacteria Plasmids as vectors Vectors are shuttle molecules that permit effective transfer and propagation in bacteria of the piece of DNA (to be cloned). Plasmids are extrachromosomal self-replicating DNA molecules. DNA could be inserted into the multiple cloning site (polylinker) of plasmid vector. Adapted from Lodish et al., Molec. Cell Biol. WH Freeman.

12 DNA CLONING in bacteria Ligation of DNA insert into the plasmid vector Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

13 Ligation of sticky ends Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

14 DNA CLONING in bacteria Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

15 DNA CLONING in bacteria Transformation of plasmids into bacteria Adapted from Lodish et al., Molec. Cell Biol. WH Freeman Animation clip: Plasmid Cloning

16 Polymerase Chain Reaction (PCR) Amplification of a DNA region Animation clip PCR Exe Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

17 Designing oligonucleotide primers 5 3 Region to amplify across CACAGACTCAGAGAGAACCCACC Forward primer 5 AGACTCAGAGAGAACCC3 3 GTGTCTGAGTCTCTCTTGGGTGG TACCCCCGTGGTCTTTGAATAAA 3 3 GGGGCACCAGAAACTTA5 Reverse primer ATGGGGGCACCAGAAACTTATTT 5

18 Designing oligonucleotide primers How long should primers be? Too short might hybridize to non-target sites Too long increases time needed for hybridization If total human DNA and 8-mer primers are used for a PCR experiment, a number of different fragments will be amplified. Occurrence of primer recognition sequence: once every 4 8 = bp ~46,000 possible sites in 3x10 9 bp of nucleotide sequence. A good length for primers should not be less than 17 nucleotides (in practice: 18-25). Occurrence of primer recognition sequence: once every 4 17 = 1.7 x bp

19 Designing oligonucleotide primers Another consideration Melting temperature (T m ) Denaturation temperature Annealing/hybridization temp Extension temperature = 94 o C = depends on T m of primers = 74 o C (optimum for Taq polymerase) Annealing temperature is very important. Affects specificity of rxn. If temp too low mismatched hybrids If temp too high no hybridization takes place Ideal annealing temp must be low enough to allow hybridization and high enough to prevent mismatched hybrids.

20 Designing oligonucleotide primers Calculating T m The Wallace Rule: Empirical and convenient. For perfect-matched duplexes, nucleotides in length T m ( o C) = 2(A+T) + 4(G+C) where (A+T) is the sum of A & T residues, (G+C) the sum of G & C residues in the oligonucleotide Bolton and McCarthy: For lengths <100 nucleotides, [Cation] < 0.5M GC content (30-70%) T m ( o C) = 81.5 o C (log 10 [Na + ]) (%[G+C]) 675/n 1.0m where n = no. of bases in oligonucleotide, m = %base-pair mismatches

21 Exercise: How would you design oligonucleotide primers (about 20-mer) that would enable you to: (a) amplify the underlined region, (b) and then clone this into an EcoRI (GAATTC) site in a plasmid? 5 cacagtaacctcaactcctgccacaatgtacaggatgcaactcctgtcttgcattgcactaagt cttgcacttgtcacaaacagtgcacctacttcaagttctacaaagaaaacacagctacaactggagc atttactgcgctaccgttcgatggtctagactggctac

22 Constructing DNA Libraries What is a DNA library? A DNA library is a collection of DNA clones in a vector. Genomic DNA library cdna library A genomic library can be screened for clones containing a sequence of interest. Cloning entire genomic DNA of higher organisms into plasmid vectors is not practical: relatively low transformation efficiency of E. coli the small number of transformed colonies that can be grown on a typical culture plate. Bacteriophage/Cosmid vectors are better, as they do not suffer from such limitations. Plasmids (insert size: ~4 kb); Bacteriophage (insert: 20 kb); Cosmids (insert: 45 kb)

23 Constructing cdna (complementary DNA) Libraries Generating cdna from mrna (using reverse transcriptase) From mrna i.e. no introns

24 Constructing DNA Libraries in bacteriophage λ vector Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

25 Constructing DNA Libraries in cosmid vector Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

26 Screening DNA Library to identifying a specific clone screen library by hybridization with radioactively labeled DNA or RNA probes Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

27 Screening DNA Library with oligo. probe For e.g., we can screen DNA library to identify clone carrying sequences that encode a protein of interest. Design oligonucleotide probe based on partial protein sequence All 48 (2x3x2x1x2x2) of the 20-mer probes must be synthesized Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

28 DNA Sequencing Sanger s Dideoxy-Chain termination Animation clip: Cycle Sequencing Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

29 Southern blotting (Named after Ed Southern, Oxford University) A specific DNA sequence (e.g. isolated by cloning), can be used as a probe, Animation clip Southern Blotting to detect complementary nucleic acids in complex mixtures including total cellular DNA (and RNA, in which case, is known as Northern Blotting) Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

30 Northern blotting To detect specific mrnas To study expression or activity of a specific gene Adapted from Lodish et al., Molec. Cell Biol. WH Freeman

31 DNA Microarrays To study expression or activities of a large number of genes in a high throughput manner E.g. Comparative analyses of gene expression in response to an external stimulus, in development or in cancer progression. Likened to Reverse Northern Blot RNA cdna

32 Growth of yeast cells deplete Glucose in growth media Adapted from DeRisi, et al., 1997, Science 278:680 and Lodish et al., Molec. Cell Biol. WH Freeman

33 Exploring the metabolic and genetic control of gene expression on a genomic scale in yeast using microarrays. Adapted from DeRisi, et al., 1997, Science 278:680

34 Changes in yeast gene expression as glucose level falls Adapted from DeRisi, et al., 1997, Science 278:680 and Lodish et al., Molec. Cell Biol. WH Freeman

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