Recombinant DNA and Restriction Enzymes

• Techniques used to manipulate nucleic acids • Manipulate DNA and RNA using: natural biochemical processes (e.g. enzymes), natural properties of microorganisms, biochemistry, microbiology

• CUT and PASTE (splice) particular sequences • CLONE sequences: make 10n identical copies (produce) • ENGINEER sequences: study functions, create new functions, new products • SEQUENCE DNA and RNA fragments • LOCATE sequences /fragments using ‘probe’ hybridisation

• Nucleotides (nt): e.g. a gene 500 nt in length • Bases (b): e.g. a sequencing read 400 bases long • Base pairs (bp): e.g. a genome size 3.4 Gbp in size • Average molecular weight: 330 Da/nt, 660 Da/bp

• Cheap and easy: manipulate, contain • Exponential clonal growth: 1-2-4-8-16, etc, agar, suspension • Yield limited by: available nutrients, available space • Naturally move DNA • Natural plasmid hosts and replicators • Plasmids: vectors (carriers) of DNA: manipulation, production

• Conjugation: DNA transfer • Transformation: DNA uptake • Transduction: Phage transfer

• cut vector, add DNA fragment, becomes rDNA molecule • then enters bacterium • transports into host cell • rDNA molecule multiplies • host cell divides • numerous cell divisions resulting in clone

• Exonucleases: trim from the end (5’ or 3’, or both) - Endonucleases: Cut internally

• Discovered by Luria (1950’s) • Bacteriophages infect Strain A but not Strain B • ”Restricted” to infecting strain A only

• Certain bacteria contain enzymes that ‘digest’ foreign DNA: endonucleases • Protect own DNA through chemical modification of DNA • Methylation of bases A and C • First enzymes isolated from E coli: EcoB and EcoK

• EcoK and EcoB: Cleave DNA remotely from their binding site, at random positions • 1970: Haemohpilus influenzae (HindII): fixed recognition and cleavage site

• Restriction & Methylation - Cleaves remotely

• Endonuclease OR Methylation - Cleaves at/close to recognition site

• Restriction & Methylation - Cleaves close to recognition site

• HindIII • H: genus • in: species • d: strain • III: number (order in which it was discovered)

• REs recognise, bind and cut specific sites in dsDNA • Sequence specificity per enzyme • Recognition site: 4, 6 or 8 nt long (with exceptions) • Often ‘palindromic’: In the case of restriction enzyme recognition sites: reads the same on both complementary strands of dsDNA when read in the 5’ to 3’ direction

• ‘Zipping’ together nucleic acids • Enzymes that catalyse the ATP-driven ligation • (ligate = to tie together) of nucleic acid backbones

• Depends on RES frequency in given DNA sample - Estimation is possible based on: A, G, C, and T frequency, size of RES, length of DNA fragment/genome in question

• (𝑁_𝑡𝑜𝑡𝑎𝑙) can be calculated using: 𝑁_𝑡𝑜𝑡𝑎𝑙 = 𝑥^𝑦 • y = DNA sequence length of nucleotides • 𝑥 = number of different possibilities for each nt position (A,G,C,T = 4) • e.g. six consecutive nucleotides (𝑦=6) that are equally likely to be A, C, G or T: 𝑁_𝑡𝑜𝑡𝑎𝑙=𝑥^𝑦=4^6=4096

• Each unique stretch of 𝑦 consecutive nucleotides occurs once every 𝑁_𝑡𝑜𝑡𝑎𝑙 bases • e.g. EcoRI, for which 𝑦=6 (GAATTC), occurs once every 4096 bases • The means that for a 100 kb genome, you expect to find on average: 100000/4096=24.4≈24 EcoRI sites (assuming each base is equally likely to occur)

• Determine position of RES in a DNA sample • Essential for cloning and genome analysis • Use pure DNA sample • Digest completely • Size RES fragments by electrophoresis (E/P)

• Hypothalamus and pancreas: reduce HGH, insulin, glucagon • 14 aa’s: small and simple • Known aa sequence • 5 mg purification: 500,000 sheep brains • E. coli production: unlimited (Itakura, 1977)

• Pure DNA • Host organism • DNA vector (plasmid, virus) • Appropriate promoter, etc • Restriction enzymes • Ligase • Recombinant host selection method

• Obtain insert and vector • Cut with appropriate RE • Ligate into vector • Transform into host • Isolate recombinant(s) • Purify DNA or TU product: RNA, Protein