Recombinant DNA technology entails modifying the genomes of organisms.

Things needed:

mutagen – anything physical or chemical that causes mutations in an organism

reverse transcriptase - these enzymes were discovered from retrovirses and its effects on DNA. Using reverse transcriptase, scientists are able to make DNA out of RNA.  These DNA are called complementary DNA or cDNA. After making cDNA from eukaryotic mRNA, scientists are able to input cDNA into the genome of the prokaryotic DNA. One main reason why this is useful is that prokaryotes are not able to excise or remove the introns (junk DNA or noncoding sequences) of DNA in eukaryotes. Since eukaryotes mRNA are already processed and had removed the introns, the newly made cDNA would also be free from introns!

synthetic nucleic acid – these guys are DNA and RNA produced in vitro, meaning outside of the human cell. Of course, nucleic acids (DNA and RNA) are made within the cell too!

Usages of Synthetic NA:

  1. building up the genetic code – figure out various nucleotide sequences and what it expresses
  2. create a gene that codes for a specific protein (of course you got to know to genetic code first in order to create a gene that gives you a functional protein)
  3. making DNA and RNA probe – probes are used in many biotech techniques, like the Northern and Southern blots and DNA sequencing. These probes are usually UV or florescent dyed, complimentary to the specific DNA or RNA sequence extracted from an organism.
  4. Making antisense nucleic acid molecules – antisense are nucleotide sequences that bind to DNA and RNA molecules

Restriction enzymes – these restriction enzymes are found in bacterial cells and are strictly cutting DNA only a specific points at restriction sites which are usually palindromic

**note – bacterial cells protect their DNA by methylating some of the nucleotides restriction enzymes cannot see nucleotides that are methylated

**notesticky ends and blunt ends:

EcoRI and EcoRII, are restriction enzymes that cut the site of the DNA, staggered, and also able to reanneal with the enzyme ligase. The ends are only able to anneal with its complementary DNA strand cut by the same restriction enzyme.

Adopted from Textbook: Microbiology with Disease by Taxonomy 2nd Ed. Robert Bauman

Staggered cut at specific sequence, dependent on restriction enzyme

HindII and Sma1 cut both DNA at the same point, therefore creating blunt ends. These ends are nonspecific and thus can combine with other ends.

Cuts DNA strand bluntly

vectors – are nucleic acid molecules that can carry the gene of interest into its genome such as viral genome, transposons, and plasmids.

  • small and easier to manipulate in lab
  • survives in cell, plasmids for instance are good DNA vectors because they are stable
  • recognized genetic marker – with a marker, such as ampicillin resistant, researchers can screen out the vector containing the gene of interest
  • ensure genetic expression – contain promoters

Gene Libraries – easily accessible known gene of interest in bacterial or phage clones that you have stored from previous recombinant DNA techniques