Recombinant DNA Technology I Lodish Chapters 5.2 …it all began with the discovery of the bacterial “defense” system that RESTRICTS phage growth. In the late 1960’s, Stewart Linn and Werner Arber discovered two classes of enzymes: methylases and RESTRICTION endonucleases. At the same time, Charles Richardson had purified DNA ligase of the E.coli phage T4…….all you needed to do was to “cut and ligate”.. And that’s what Paul Berg did in the 70’s…and he received the Nobel Prize in 1980.

Restriction enzymes cut DNA molecules at specific sequences

Most restriction enzymes recognize short palindromes and cut unmethylated DNA Frequency of 6 cutters: 46 = once every 4096 bp Frequency of 4 cutters: 44 = once every 256 bp

Today we know more than 600 different restriction endonucleases

DNA cloning with plasmid vectors Recombinant DNA technology depends on the ability to produce large numbers of identical DNA molecules (clones) Clones are typically generated by placing a DNA fragment of interest into a vector DNA molecule, which can replicate in a host cell When a single vector containing a single DNA fragment is introduced into a host cell, large numbers of the fragment are reproduced along with the vector

Plasmid vectors have an ori, a resistance marker and a multi-cloning site (polylinker)

Cloning is a 2 step process: 1) integrate DNA fragment into vector…

…and 2) transform E.coli to multiply DNA

Identical E.coli clones carry identical (cloned) DNA Usually great to clone short fragment of a few kb

Complementary DNA (cDNA) libraries are prepared from isolated mRNAs

Preparation of a cDNA library

How do you identify a clone that carries a specific gene?

How will I obtain the sequence of my cDNA? The Sanger (dideoxy) method

Sample printout from an automated sequencer N = nucleotide cannot be assigned

Polymerase chain reaction: an alternative to cloning The polymerase chain reaction (PCR) can be used to amplify rare specific DNA sequences from a complex mixture when the ends of the sequence are known PCR comes in two flavors: 1) DNA template based, or 2) RNA template based (reverse transcriptase PCR) ….Kary Mullis, surfing father of PCR sold the technology to Cetus for $10,000. Cetus sold the technology for a stunning $300,000,000 a few years later… Mullis received the Nobel Prize in 1993 and turned his back on both academia and industry.

Polymerase chain reaction thermostable polymerase

PCR products can be cloned into vectors (e.g., for protein expression)

Reverse transcriptase PCR (can be used in clinic to probe patient sample for oral pathogens) Start with single stranded template (mRNA) RNase H gene-specific primer add second gene-specific primer to amplify (by regular PCR)

Transposon mutagenesis This strategy is widely used to create mutants in bacteria and allows for easy identification of the disrupted gene.

Definition of recombinant DNA (artificially ligated DNA fragment) What you need to know: Definition of recombinant DNA (artificially ligated DNA fragment) Purpose of restriction enzymes (cut DNA, naturally used in defense against phages) Definition of cloning (making lots of identical organisms/DNAs) Requirements for cloning vectors (3: ori, selection marker, polylinker) Principal of DNA sequencing (use ddNTPs to terminate chain extension) Principal of PCR (use thermostable polymerase and specific primers to amplify a DNA fragment in multiple rounds of strand melting, primer annealing, primer extension). Principle of transposon mutagenesis. When to use PCR vs traditional cloning: DNA sample is limited some sequence information is available (there are thousands of bugs that have NOT been sequenced but play a role in human pathologies). in general, it is cheaper to create large amounts of recomb. DNA by cloning, and bacterial enzymes have proofreading activities (PCR can create errors) Why would you want to know about cDNA cloning? gives you information on what sequences are expressed (transcribed and translated) rather than just encoded in the genome many drugs (e.g., insulin) are produced recombinantly (from cloned sequences)