Cloning a DNA segment from lambda bacteriophage

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 bacteriophageRecombinant DNA technologyFirst recombinant protein used as drug - insulin from E.coli (1982)



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 100-1000 base pairs apart so that the restriction fragments are >100 bp long



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



Part 1 of labDigestion of lambda bacteriophage DNA with EcoRIDigestion of pUC18 plasmid DNA with EcoRI

Other facts about restriction endonucleasesamount 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 bufferjust 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 MgCl2, 50 mM Tris-HCl pH 8)

enzymes come from company in a concentrated form, stored at -20 ˚C in buffer with 50% glycerolreason for glycerol storage - does not freezefreeze-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 bacteriophageDNA ligation into plasmid

Ligate small DNA fragments into plasmid DNAPlasmid has single, unique EcoRI site

Cloning a DNA segment from lambda bacteriophageDNA ligation into plasmid

Ligate small DNA fragments into plasmid DNALambda bacteriophage has many EcoRI sites

Restriction map of lambda DNA - EcoRI

21,226 4878 5643 7421 5804 3530

21,226

Sizes of restriction fragments

Position of EcoRI restriction sites

26,104 31,747 39,168 44,972


DNA ligation into plasmid

Ligate small DNA fragments into plasmid DNA

DNA was cut with EcoRI and pUC plasmid has been cut with EcoRI


DNA ligation into plasmidLigation by DNA Ligase - in cell, ligase used in DNA replication, repair, recombinationT4 DNA Ligase - isolated from T4 phage infected E.coli (requires Mg2+ and ATP)


DNA ligation into plasmidLigation ATP

Positively charged

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Cloning a DNA segment from lambda bacteriophage