Some Molecular Biology Techniques 1

Some molecular things Restriction Enzyme digestion Ligation of DNA Cloning DNA PCR cDNA synthesis Gel electrophoresis Southern /Northern /Western Blot Sequencing 2

Restriction Enzyme digestion: Restriction endonucleases (3,000 studied) 600 commercially available Cut double stranded DNA at recognized base pairs First found from microbes Usually named after the organism eg. E.coli I (Escherichia coli), Sal I ( Streptomyces albus ), Sma I ( S erratia marcescens ) EcoRI 5'GAATTC Sma I 5'CCCGGGEcoRI 3'CTTAAG 3'GGGCCC –5'---G A ATTC---3' 5'---CCC GGG---3’ –3'---CTTA A G---5' 3'---GGG CCC---5' “Blunt” end restriction enzyme“sticky” end restriction enzyme 3

Ligation of DNA: Ligase = an enzyme that join double strand DNA pieces together Catalyze the sugar phosphate backbone of the DNA together T 4 DNA ligase ATP dependent 37°C 4

Cloning DNA More than one = library Any DNA can be cloned EST = Express Sequence Tags cDNA = Copy DNA from transcriptome 5

PCR-based markers Polymerase Chain Reaction amplification of tracts of DNA defined by border sequences that hybridize to selected DNA polymerase primers Requires: - target DNA - thermostable DNA polymerase (Taq) - oligonucleotide primer(s) (7-30nt) - dNTPs + Mg++ 6

PCR Considerations Does not require high quality DNA Extremely sensitive - easily contaminated Reaction conditions must be optimized and controlled Fast Relatively inexpensive “amplicon” + PCR primer oligos 7

Primer design Primer length to be considered (18-24 bases) –Exceptions (RAPD = 10-12, Oligomicroarray = 80) Melting temperature (50°C to 60°C) T m ( o K)={ΔH/ ΔS + R ln(C) Primer annealing temperature T a = 0.3 x T m (primer) T m (product) – 14.9 GC content (40 to 60%) Secondary structure (avoid repeat within primer <4 repeat eg. ATAT) Runs (avoid stretches of CCC or GGG etc.) End in “G” if possible for more stability 8

Polymerase Chain Reaction: Diagram of the first 2 cycles /ph_tour/pcr.fgsr 9

Gel Electrophoresis: Use for multiple purposes: –Check restriction digestion –Check ligation efficiency –PCR products –Clone sizing 10

Reverse transcriptase: an RNA-dependent DNA polymerase 11

Blotting: Southern /Northern /Western Blot 12

G A A T T C T G G C A T C G T T A A C G G T A T C G A T C  Method invented by Frederic Sanger in 1977  Method still used today  Can be automated to sequence large stretches of DNA rapidly  Can be used to sequence Genomes Dideoxy DNA Sequencing S. Chowrira 13

G A A T T C T G G C A T C G T T A A C G G T A T C G A T C The sequencing method developed by Fred Sanger forms the basis of automated "cycle" sequencing reactions today. This method uses chemically altered "dideoxy" bases to terminate newly synthesized DNA fragments at specific bases (either A, C, T, or G). These fragments are then size-separated, and the DNA sequence is deduced. A laser within an automated DNA sequencing machine analyzes the DNA fragments produced. Fluorescent dyes are added to the reactions 14

Sanger autosequencers 15

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Start of automation? 17

Bioinformatics Software to “read” the signals based on peak intensities from the florescense signals eg. Hex, Tamara, Cy3, Cy5, Infrad 700, Infrad 800 Beware of background and noise Beware of double or more peaks Peak quality assessment 18

At last some sequence data ATGCATGAGNTA 19

More sequences…… ATGCATGAGNTAAGT GAGTATACCGATANA GAACTGCGCAGTCGA CGGGCCTTTACGACG ACGCGCTACGCACGT ATGCAATTGGCCATG ACAGTCGACGACACG TAGACGCCCAGGCA GCTGACGACTGCGTG 20

1 agtaggatgc ggccagcgca ggagctggtg gtgaaaaact gatctcgcat gttcagaaac 61 tgcacaagct ctaccacatc ctcgtcaaca ctgcccttcc ggctgaggtc cgctttgctc 121 aaacattgcg ccttccattt cctgaactcc gcgctgcgat ccatgggtga cggactcagg 181 gtctgcgctc ggctccttca tggtagcctc gtctctccct gggggcagcc atattgaagc 241 gagcggtggc cgaaaaggtc caaacttcct cttcgttctt aatttgggaa attctagtgc 301 cttcgcctag gggtggagag aggcgcacat cctgggtgga gcgaggcgca catcctgggt 361 ggagcctgca aaagttggag aaggtgtggc agtcctcatt tctgtggaat ctgaagaagc 421 ccacgcagat tttaattccc actctagatt ctggttctaa tcaaggtcca tgcatgtggt 481 gtcaaccccg tggagacata cattcgctct ggtacttata gtagaaaacc actcttaccc 541 tatactcctg gctcagatgt ggctggggtg atagaagctg ttggagataa tgcatctgct 601 ttcaagtgcc tgtgtgaaag ctggagagag tgttctggtt catggggcaa gtggaggagt 661 tggattagca gcatgccaaa ttgctagagc ttatggctta aagattttgg gcactgctgg 721 tactgaggaa ggacaaaaga ttgttttgca aaatggagcc catgaagtgt tcaatcacag 781 agaagtgaat tacattgata aaattaagaa gtatgttggt gagaaaggaa ttgatataat 841 tattgaaatg ttagctaatg taaatcttag taaagacttg agtcttctgt cacatggagg 901 acgagtgata gttgttggca gcagaggtac tattgaaata aacccacgag acaccatggc 961 aaaggagtcg agtataattg gagttactct cttttcctca accaaggagg aatttcagca 1021 atatgcagca gcccttcaag ctggaatgga aattggctgg ttgaaacctg tgataggttc 1081 tcaatatcca ttggagaagg tggccgaggc tcatgaaaat atcattcatg gtagtggggc 1141 tactggaaaa atgattcttc tcttatgatg attaattctt tcatggattt cctatgtaat 1201 tagaggtact gtctttcccc cagttgtact taccctatct tttctttaat taacattcga 1261 ttccatgagc ttcttatgtg aaaaaataag atttttcttt agagagcaga agcagaagag 1321 taaaatttat tgtatagcta gcaatatttt tttatgccat ctgtctcaaa tcaaagagtc 1381 atcatagtag gaaataacat gttagttgtc atttggcatg agtgtgcatt ccagtaattc 1441 ttaattgata tttgattaat tccatacctt tgattaaaac atgctagttc aaaataagac 1501 tgctcagttt ccaagggttt tcaagcctac ttacctttat aaaggttctc tagtctctga 1561 ttagccatga ctgtattgga ctttgaacat tttctgaact aaaaacctct attctaaact 1621 aatctcattt ggatgtgtaa gtcttttgta aaggcaagaa taaataatat ccaggacaat 1681 ttattagttt tctcagtatt ttcccaaata ttagaatatt tacttcatta ttggttggct 1741 gccaatgacc ccatatgttc tgtgagaata gtagctttat ctttgatata atacatagtc 1801 tccaaatagg taatacttcg caattgatta gattttcaga gtagatttag agttatctgt 1861 ttttctggtg agggtcaaat atttttgtta attaagctca caaatttgat aaattaagaa 1921 ttatctgcat ttgtctcgta acataataat gtgtaataaa gtctatagaa aattaaaaaa 1981 aaaaaaaaaa Endless sequences….Oh Boy! Now What! 21