GENE TECHNOLOGIES

• Genetic code= universal meaning all has AT CG as bases
• Universal code- means that codons code for the same AAs in different organisms so dna codons can be transferred from one organism into the gene sequence of another
• Dna that’s been transferred between organisms is recombinant dna and organisms with recombinant dna are transgenic
• Transcription and translation are also universal
• Transgenic organisms or GMOs are made by inserting dna fragments from one organism  into the nucleotides of another
• The steps to creating a GMO/transgenic organism...​

  1. Isolation of desired DNA fragment​
  2. Multiplication of desired fragment using PCR- polymerase chain reaction​
  3. Transfer into the organism using a vector such as plasmids liposomes or viruses​
  4. Identification of the desired fragments using a marker such as genes that code for identifiable characteristics like colours. The fragments are then cloned
• Ways to extract fragments...

   RESTRICTION ENDONUCLEASE-extract the desired gene fragment from donor dna​

  • Enzyme which cuts off the desired gene from a dna strand​
  • It cuts the phosphate-sugar ends unevenly to leave blunt and sticky ends​
  • Sticky ends are longer than blunt ends and make it easier for the fragment to form hydrogen bonds with complementary sticky ends on different strands​
  • Sticky ends just mean ends of dna fragments that overhang and have exposed nucleotides
• Ways to extract fragments...

  REVERSE TRANSCRIPTASE- to synthesis a complementary strands of cDNA based on donor mRNA​

  • MRNA  is isolated and cDNA is made by reverse transcriptase​
  • Dna polymerase then creates a double stranded DNA molecule based off the cDNA​
  • No introns!!!!
• Ways to extract fragments...

  GENE MACHINE- artificially synthesising the gene by putting nucleotides in a gene machine​

  • Bc scientist know our proteome genes can be sequenced artificially​
  • Computers instead of mrna are used to synthesise gene sequences​
  • Short dna fragments are produced and then joined with other short fragments to make long nucleotide sequences​
  • The long sequences are then inserted into vectors such as plasmids​
• THE PCR

  • USED TO AMPLIFY FRAGMENTS OF DNA ​
  • REPEATED OVER AND OVER​
  • EACH CYCLE DOUBLE AMOUNTS OF DNA
• THE PCR PROCESS

  1. DNA sample. Primers, free nucleotides and DNA polymerase are heated to 95C to break the hydrogen bonds between the double helix​
  2. The mixture is cooled to 50-65C so the primers can bind/anneal to the strands​
  3. The mix is heated again to 72C- optimal for  DNA polymerase ​
  4. Free nucleotides line up with complimentary free nucleotides and form strands via DNA polymerase​
  5. TWO NEW COPIES of the fragment DNA are formed. THIS IS ONE CYCLE OF PCR​
  6. The cycle starts again
• • Not all of the genome codes for proteins​
  • Some have repeating base sequences that don’t code for anything- these are called VARIABLE NUMBER TANDEM REPEATS and everyone has different ones in different places and amounts
• GENETIC FINGERPRINTING PROCESS

  1. A sample of DNA is obtained​
  2. PCR makes copied of the VNTRs and primers are placed either side​
  3. A fluorescent tag is added to the amplified DNA fragments​
  4. The DNA fragments undergo gel electrophesis​
  5. The DNA fragments are viewed as bands under UV light
• GEL ELECTROPHESIS​

  1. The DNA mixture is placed into a well in a slab of gel and covered in a conductive buffer​
  2. Electrical currents are passed through the gel​
  3. DNA fragments are negatively  charged so move towards the positive electrode. ​
  4. Small fragments move faster and travel further so fragments are seperated by size​
• • DNA probes are used to locate specific alleles ​
  • This can be used to find out if someone has a genetic disorder or is a carrier like CF
• • The probe will bind to a specific allele​
  • The probe also has a radioactive of fluorescent marker
• GETTING GENETIC PROBES

  1. A sample of DNA ia combined with restriction enzymes and seperated using electrophesis​
  2. The seperated fragments are transferred to a nylon membrane and incubated with the labelled probe​
  3. If the allele is present the probe will bind to it​
  4. The membrane is exposed to uv light to see if the gene is present. If it is there will be a fluorescent band
• GETTING GENETIC PROBES

  1. A DNA microarray is a glass side with microscopic spots of DNA probes attatched in rows​
  2. A sample of labelled DNA is wasged over the array​
  3. If a specific gene is present then the probes will attach and any labelled dna will show up under UV light​
  4. Any spot that fluoresces has a specific allele.​
  5. ​

  THIS METHOD SCREENS LOTS OF GENES AT ONCE
• Ligase joins fragments of DNA together
• Transgenic organisms are used to produce drugs, vaccines, biofuels, industrial enzymes, and bioremediation
• The host cell produces large quantities of the protein encoded by the gene using its own machinery.
• Recombinant dna technology involves isolating DNA fragments containing genes coding for desired products (e.g. insulin) from an organism and inserting them into plasmids which are then inserted into bacteria or yeast cells.