L1-4 gene biotech

• biotechnology 


  manipulation of living organisms or their components to produce useful products
• central dogma of molecular biology

  DNA to RNA to protein only, cannot get back to RNA from protein, however can get back to DNA from RNA using reverse transcriptase
• cDNA 


  synthesised from mRNA using reverse transcriptase

  requires primers and nucelotides (dATP, dGTP, dCTP and dTTP)

  produces single stranded cDNA
• complementary DNA 


  second strand is synthesised using primers, nucleotides and enzyme (DNA polymerase)
• cDNA doesn't contain intron genes
• gene cloning

  • DNA inserted to plasmid (produce recombinant DNA molecule)
  • vector transports gene to host
  • vector multiplies in host
• plasmid is small, circular DNA can be replicated independently in bacteria from chromosomal DNA
• 3 important features of plasmid

  • origin of replication
  • selectable marker (such as bacterial resistance)
  • cloning/restriction enzyme cleavage site (DNA sequence that is recognised and cut by restriction endonuclease)
• restriction enzymes 2 types

  • blunt ends
  • sticky ends (complementary bases on each end)
• restriction ends leave phosphate on 5' end and OH on 3' end
• DNA ligase joins 2 fragments, which ligate phosphodiester backbone
• DNA ligase joins 5' phosphate to 3' hydroxyl in ds DNA
• host must be non-pathogenic to work in the labs
• competent species

  • treatment that enhances their ability to take up DNA
• not all plasmids taken up by host cells, during transformation
• tell which have transformed

  • antibiotic resistance on plasmid
  • colonies remaining have taken up the plasmid
• issues with transformation

  • self ligated vector molecule
  • wrong recombinant DNA
  • desired recombinant
• phosphatase used to remove phosphate from plasmid so only recombinant will bind back together
• enzyme beta-galactosidase converts the substrate X-gal into blue product
• blue fragments contain the gene on the plasmid and are used to transform the bacteria
• gene library


  collection of clone sufficient in number to be likely to contain every single gene present in a particular organism
• enzyme commonly used to insert DNA into plasmids during cloning

  DNA ligase
• what is a selectable marker in plasmids used for in DNA cloning?

  identification of transformed cells
• in white-blue screening system, you will obtain blue colonies if

  B-galactosidase is expressed, DNA has not been ligated into the plasmid
• blue colony is non-recombinant and white colony is recombinant
• agarose gel electrophoresis function

  separate DNA molecules based on size
• nucleic acids are negatively charged so migrate to positive electrode
• composition of the gel determines the sizes of molecules that can be separated
• increase % of agarose, smaller molecules get through larger don't
• techniques to study gene/DNA structure

  • restriction mapping
  • fragment length polymorphism
  • polymerase chain reaction
  • southern analysis
  • DNA sequencing
• constructing restriction map

  • series of restriction digests
  • number and sizes of the fragments produced by each restriction endonuclease determined by gel electrophoresis
  • information must be supplemented by series of double digestions
  • compare results of single and double digestions, allows many restriction sites to be mapped
• PCR


  is the amplification of DNA
• Taq polymerase is thermostable DNA polymerase
• PCR steps

  • denaturation of DNA at 94 degrees Celcius
  • annealing at 50-60 degrees Celcius
  • synthesis of new DNA at 74 degrees Celcius
  • repeat reaction to double number of DNA copies each time
• uses of PCR

  • amplification of desired sequence prior to cloning
  • diagnostic and forensic application
  • species identification
  • identification of disease alleles
  • gene expression
• southern blotting

  • allows specific fragments to be identified in a complex mixture
• southern blotting

  • DNA is digested with restriction endonuclease to analyse DNA
  • DNA fragments can be separated by gel electrophoresis
  • DNA denatured
  • filtered with labelled probes, bind to complementary DNA sequences
  • probe bound to filter detected by exposure to film, reveals DNA fragment
• northern transfer is for RNA molecules
• western transfer is for proteins
• dideoxynucleotides stops the DNA polymerase from adding nucleotides to the growing DNA strand