B21 recombinant DNA technology

• reverse transcriptase is an enzyme which catalyses the formation of a double strand of DNA from a single strand of RNA
• reverse transcriptase can be used to make working versions of DNA which act as genes by extracting mRNA from cells where that gene has been expressed
• restriction endonucleases are enzymes which cut DNA at specific sequences
• the most useful restriction endonucleases make staggered cuts in DNA, producing fragments which have single-stranded sticky ends
• sticky ends are useful as if the same restriction endonuclease is used to cut two DNA fragments, the ends will be complementary
• the gene machine is an automated way of manufacturing genes, by inputting the desired sequence of bases, checking it for biosafety and biosecurity, and letting the computer design small oligonucleotides which can be assembled into the whole gene
• the sequences of DNA cut by restriction endonucleases are called recognition sites
• if recognition sites are cut in a staggered way, each end has a single strand which is a few nucleotide bases long, this is called a sticky end
• if the same restriction endonuclease is used to cut all the DNA to produce sticky ends, all the fragments will be complementary to each other
• once the complementary bases of two fragments with sticky ends have paired up, an enzyme called DNA ligase binds the fragments together by forming phosphodiester bonds
• the molecule produced when the complementary bases of two fragments with sticky ends have paired up, and DNA ligase binds the complementary strands together, is called recombinant DNA
• recombinant DNA needs to have more DNA added to it:

  • promoter region
  • terminator region
• a promoter region is the binding site for RNA polymerase, required for synthesis of mRNA and therefore transcription of the DNA fragment
• a terminator region is the release point for RNA polymerase, required to stop transcription at the correct time
• once the DNA fragment is cut out, and has had promoter and terminator regions added to it, it needs to be joined into a carrying unit called a vector
• there are many types of vector but the most common is a plasmid, a circular length of DNA found in bacteria, separate from main bacterial DNA
• the polymerase chain reaction is an automated method of copying fragments of DNA
• the PCR requires:

  • the DNA fragment which is to be copied
  • DNA polymerase
  • primers
  • nucleotides
  • thermocycler
  • buffer solution
• primers are short sequences of nucleotides which have a set of bases complementary to those at one end of each of the DNA fragments
• a thermocycler is a computer-controlled machine that varies temperatures precisely over a period of time
• the PCR has three steps:

  • separation of the DNA strand
  • annealing of the primers
  • synthesis of DNA
• in PCR, in separation of the DNA strand:

  • DNA sample, primers, free nucleotides and DNA polymerase are added to a vessel in the thermocycler
  • mixture is heated to 95 degrees to break the hydrogen bonds and separate the two strands
  • this denatures the DNA
• in PCR, in annealing of the primers:

  • mixture is cooled to 55 degrees
  • primers bind to the strands
  • this prevents the separate strands from rejoining and provides the starting sequence for DNA polymerase to attach nucleotides to
• in PCR, in synthesis of DNA:

  • mixture is heated to 72 degrees
  • DNA polymerase binds complementary nucleotides along each of the DNA strands
  • this produces two identical strands of DNA
• in vitro cloning means using the polymerase chain reaction

  in vivo cloning means transferring the fragments to a host cell using a vector
• advantages of in vitro cloning:

  • extremely rapid
  • does not require living cells
• advantages of in vivo cloning:

  • can introduce a gene into another organism
  • almost no risk of contamination
  • very accurate
  • cuts out specific genes
  • produces transformed bacteria which can be used to produce large quantities of useful products
• a DNA probe is a short, single-stranded length of DNA that has some sort of label attached which makes it easily identifiable, designed to be complementary to a specific sequence
• the most common types of DNA probe are:

  • fluorescently labelled, can be detected using UV light
  • radioactively labelles, can be detected using x-ray film
• DNA probes are used to identify particular alleles by:

  • a probe is made which has a complementary base sequence to the base sequence of the allele
  • the double-stranded DNA that is being tested is treated to separate it into two strands
  • the separated DNA strands are mixed with the probe
  • the probe binds to the complementary sequence if it is present
  • the sample is viewed using x-ray film or UV light depending on the labelling of the probe
• DNA hybridisation is the process by which the DNA sample is combined with the complementary single-stranded probe
• for DNA hybridisation to take place, the two strands of the DNA molecule must be separated, this is done by heating the DNA until the hydrogen bonds between the strands break, this is denaturation
• in DNA hybridisation, as the mixture cools, the single-stranded DNA will reform hydrogen bonds, either with the complementary DNA strand, or with the complementary probe, this is annealing
• to determine whether someone possesses a specific allele, the whole process is:

  • determine the base sequence of the allele
  • produce a fragment of DNA with the complementary base sequence
  • amplify the fragment using PCR
  • attach a marker to create a DNA probe
  • extract DNA from the person
  • denature the DNA
  • anneal the DNA in a mixture containing the DNA probe
  • wash out the vessel containing the sample
  • test using x-ray film or UV light
• genetic screening can be used to determine the probabilities of individuals having offspring with genetic disorders, to detect oncogenes, and to develop personalised medicine such as correct dosage
• genetic counselling is when advice and information about genetics is given to people in order for them to make decisions about themselves and their potential offspring
• genetic fingerprinting is a technique which can detect differences in people’s DNA
• VNTRs are variable number tandem repeats, short repeating sequences in the genetic code
• the chance of two individuals having identical VNTRs is extremely low, and the more closely related two individuals are, the more similar the VNTRs will be
• gel electrophoresis is a process used to separate the DNA fragments and proteins according to their size using an electric current