Recombinant technology

Recombinant technology

1. Standard DNA cloning

   • Restriction enzymes cut DNA into manageable size (restriction fragments)
     • Restriction enzymes usually:
       • act as dimers and recognize short palindromic DNA sequence
       • have precise recognition sequences
       • some leave overhangs, others cut the DNA flush (in the middle) and are called blunt restriction enzymes
     • DNA is digested in a small volume of buffered water at 37C for 2 hours
       • Restriction enzymes are identified and purified from bacteria -
         • They have evolved to stop entry of foreign DNA into the cell.
   • Restriction fragments can be separated from each other by gel electrophoresis
     • DNA is negatively charged - so it is attracted to the anode which is positive.
       • Think of the gel (matrix) as a thick forest and the DNA as different sized dogs: The smaller dogs can find their way through the tangle of under brush faster than the larger ones.
         • We use dyes like ethidium bromide to stain the DNA, we then purify the DNA from a small slice of the gel
   • Cloning DNA involves ligation of two DNA fragments
     • Ligase is an enzyme that joins DNA fragments to create recombinant DNA
       • Blunt fragments ligate together, but not very well
       • Cohesive termini (“sticky ends”) ligate well because they can hybridize:
   • Cloning DNA usually involves ligation of DNA fragments into a plasmid vectors
     • Plasmids are small, circular, extra-chromosomal DNA that occur naturally in bacteria.
       • They have their own origin of replication that usually results in about 50 copies of the plasmid being made in each bacteria.
         • They usually carry antibiotic resistance genes.
     • Plasmid vectors are made from plasmids, usually by adding a bunch of restriction enzyme sites in one part of the plasmid (called a multiple cloning site)
       • Plasmid vectors only hold <30 kilobases of DNA
         • Bacterial artificial chromosome (<300 kilobases)
         • Yeast artificial chromosome (<3 megabases)
         • Vectors can be bought as purified DNA from many companies
   • Cloning DNA involves transformation (putting the DNA into bacteria)
     • Transformation involves mixing bacteria with the plasmid DNA and creating temporary holes in the cell membrane (electroporation or chemical treatment)
       • Competent bacteria are bacteria that are ready to take up new DNA.
         • This process is not very efficient, so the treated bacteria are selected on antibiotic plates.
           • Colonies grow that contain thousands of bacteria, that each contain 50 plasmids that all originated from a single plasmid. That is why its called cloning!
             • Single colonies are lifted from the plate to start a liquid culture, the plasmid can be easily purified from the bacteria and stored or analysed.

2. Where do we get the DNA to start with?

   • by making a library of cDNA clones from mRNA - only contains things that are expressed
     • Advantage: One approach to studying disease is to identify which genes are expressed in diseased tissue - then compare to normal tissue. This may identify the genes involved with the disease.
     • Cloning cDNA:
     • cDNA library:
       • To isolate single clones from the mixed population it is important that there is only:
         • - one insert in each plasmid,
         • - one plasmid in each bacteria,
         • - one bacteria starts each bacterial culture
       • Some clones will be house keeping genes and others will be tissue specific
       • Highly transcribed genes will be cloned more often, unfortunately we are more interested in the regulatory genes that are probably very rare
     • Things one can do with a cDNA library:
       • sequence the ends of all of the clones in the library - called expressed sequence tags (ESTs)
       • now that sequencing is inexpensive, we often sequence entire libraries. Many companies have lists of genes that they have cloned.
       • use for genetic engineering
       • use for microarray analysis
   • by making a library of genomic clones - contains everything
     • Advantage: Contains all the regulatory sequences, allowing one to study transcriptional regulation
     • Genomic Library:
       • What is a genomic library useful for?
         • Sequencing genomes!!!
         • Identifying regulatory sequences.

3. Dideoxy terminator sequencing:

   • To sequence we first denature (100˚C) our template so that it is single stranded. Then we allow it to cool with the primer. Then we start a DNA synthesis reaction with DNA polymerase and NTPs
     • Primers (also called oligomers) are short (~20nt) single stranded DNAs that can be easily synthesized. We often design them to anneal to sequence on the edge of the vector.
   • Dideoxy terminator sequencing results in a separate bands on a gel:
     • Running all four ddNTP reactions on the same gel results in a nucleotide ladder
   • Automated sequencing

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