Cloning a DNA segment from lambda bacteriophage Recombinant DNA technology Allows study of the structure & function of a single protein coding gene in purified form Allows amplification and isolation of specific genes using simple procedures (usually involves help of bacterial cells and plasmids) Trying to purify a specific gene from cellular DNA would require a magnitude of purification & lots of starting material

Cloning a DNA segment from lambda bacteriophage

Recombinant DNA technology First recombinant protein used as drug - insulin from E.coli (1982)

Cloning a DNA segment from lambda bacteriophage Recombinant DNA technology To isolate segment of DNA to be cloned usually need to cleave it out of larger piece of DNA (or can PCR amplify) How do scientists do this? Use RESTRICTION ENDONUCLEASES RESTRICTION ENDONUCLEASES - protein enzymes that cleave the phosphodiester bonds that connect the nucleotide units in DNA or RNA at very SPECIFIC sites These enzymes are mainly produced by bacteria where they degrade invading foreign DNA; they have been purified from these sources and are now available commercially (300 RE available) Most restriction enzymes recognize a specific sequence of 4-6 nucleotides in DNA and each will cut the DNA into discrete pieces known as restriction fragments If a segment of DNA has more than 1 restriction site for the same enzyme, those sites are usually base pairs apart so that the restriction fragments are >100 bp long

Cloning a DNA segment from lambda bacteriophage Recombinant DNA technology Cohesive ends very important in recombinant DNA procedures because it allows two DNA fragments to be linked together by complementary base pairing at their ends

Cloning a DNA segment from lambda bacteriophage Recombinant DNA technology Part 1 of lab Digestion of lambda bacteriophage DNA with EcoRI Digestion of pUC18 plasmid DNA with EcoRI Other facts about restriction endonucleases amount of enzyme usually given in units of activity 1U is usually amount of enzyme needed to digest 1 µg of phage DNA in 1 hour at 37 ˚C in correct buffer just like all enzymes, RE have optimal reaction conditions (pH, buffer-ionic strength, temperature) EcoRI has a preference for buffers with high ionic strength (100 mM NaCl, 10 mM MgCl 2, 50 mM Tris-HCl pH 8) enzymes come from company in a concentrated form, stored at -20 ˚C in buffer with 50% glycerol reason for glycerol storage - does not freeze freeze-thaw BAD for protein enzymes DO NOT CONTAMINATE ENZYME STOCK TUBES!!!! Heat inactivate restriction enzyme (70 ˚C) Ligate cleaved fragments to each other

Cloning a DNA segment from lambda bacteriophage DNA ligation into plasmid Ligate small DNA fragments into plasmid DNA Plasmid has single, unique EcoRI site

Cloning a DNA segment from lambda bacteriophage DNA ligation into plasmid Ligate small DNA fragments into plasmid DNA Lambda bacteriophage has many EcoRI sites Restriction map of lambda DNA - EcoRI 21, ,226 Sizes of restriction fragments Position of EcoRI restriction sites 26,10431,74739,16844,972

Cloning a DNA segment from lambda bacteriophage DNA ligation into plasmid Ligate small DNA fragments into plasmid DNA DNA was cut with EcoRI and pUC plasmid has been cut with EcoRI

Cloning a DNA segment from lambda bacteriophage DNA ligation into plasmid Ligation by DNA Ligase - in cell, ligase used in DNA replication, repair, recombination T4 DNA Ligase - isolated from T4 phage infected E.coli (requires Mg 2+ and ATP)

Cloning a DNA segment from lambda bacteriophage DNA ligation into plasmid Ligation ATP Positively charged