6.1.3 - manipulating genomes

• DNA sequencing is identifying the base sequence of a DNA fragment
• sequencing used to be a manual process however now it has become automated. Entire genomes can now be read.
• benefits of genome wide comparisons:

  • comparing between species allows us to determine evolutionary relationships
  • comparing between individuals of the same species allows us to tailor medical treatment to the individuals.
• DNA sequencing can be used in synthetic biology as knowing the sequence of a gene allows us to predict the sequence of amino acids that make up its polypeptide. This allows for development of synthetic biology.
• DNA profiling is identifying the unique areas of a persons DNA in order to create a profile that individual to them.
• dna profiling is used for:

  • forensic science - DNA obtained during crime investigations can be used to identify suspects
  • medicine - to screen for a particular base sequence to identify inheritable diseases.
• we can amplify DNA fragments using the polymerase chain reaction (PCR). Makes millions of copies of a DNA fragment which are then cut at different lengths in order to be sequenced.
• the reaction mixture at the first stage of PCR contains the DNA fragment to be amplified, primers that are complementary to the start of the fragment, free nucleotides to match up to exposed bases and DNA polymerase to create the new DNA.
• PCR:

  1. heated to 95 degrees to break apart hydrogen bonds holding DNA strands
  2. cooled to 55 degrees so primers can bind
  3. heated again to 72 degrees to activate DNA polymerase (usually taq polymerase) and allow free nucleotides to join
  4. new DNA acts as template for next cycle
• electrophoresis is used in DNA profiling by

  • DNA fragments of varying lengths are placed at one end of a slab of agar gel
  • Electric current is applied so DNA fragments move towards the other end of the gel
  • shorter fragments travel further. The pattern of bands created is unique to every individual
• Genetic engineering is where a DNA fragment from one organism is inserted into the DNA of another organism sometimes acRoss different species. This is done through use of a vector and a host cell.
• To isolate a DNA fragment restriction enzymes cut DNA at specific sequences. Different restriction enzymes cut at different points but one restriction enzyme will always cut at the same sequence. Therefore using particular restriction enzyme allows you to cut out a certain gene of interest.
• to insert a DNA fragment into a vector a plasmid is used as the vector and is cut using the same restriction enzymes as the DNA so that the ends are complementary. DNA ligase joins the fragment and plasmid together.
• To insert a vector into a host cell, the host cells are mixed with the vectors in an ice-cold solution then shocked to increase the permeability of the cell membrane ( electroporation ) which encourages the cells to take up the vectors.
• arguments for and against genetic engineering :

  + insect resistance can be introduced to crops

  + genetically engineered animals used to produce pharmaceuticals ( pharming )

  + genetically engineered pathogens can be produced for research

  -genetically engineered seeds would be hard to acquire for poorer farmers
• gene therapy is replacing a faulty allele (one that codes for a genetic disease) with a normal allele.
• the two types of gene therapy are

  • somatic
  • germ line
• somatic gene therapy is when the allele is introduced to target cells. It is only short term and needs repeating.
• germ line gene therapy is when the allele is introduced to embryonic cells so it is present in all resultant cells. it is permanent as it is passed onto offspring.
• DNA sequencing: finding the nucleotide sequence for a gene or the whole genome
• Sanger sequencing works by:

  • Create copies of DNA fragments
  • create complementary strands for each fragment
  • analyse complementary fragments by gel electrophoresis
• to create copies of DNA fragments in Sanger sequencing you extract DNA, heat it to separate the strands, cut it into fragments and make many copies
• to create complementary strands for DNA fragments in sanger sequencing the copied DNA is put into a mixture with DNA nucleotides, DNA polymerase, DNA primers and terminating DNA nucleotides. DNA polymerase use dna primers to attach to fragments and then make complementary strand
• a terminator nucleotide stops any more nucleotides from being added.
• To analyse complementary fragments in Sanger sequencing the fragments are separated by length using gel electrophoresis and the original nucleotide sequence is worked out using the end base.
• high throughput sequencing is:

  • automated
  • rapid
  • cheaper than Sanger
• Benefits of DNA sequencing:

  • allows genome wide comparisons between individuals and species to reveal relatedness
  • predict amino acid sequences and tertiary structure of polypeptides
  • used for synthetic biology, modifying existing sequences to make drugs
• There are two ways a DNA fragment can be produced:

  • restriction endonuclease
  • reverse transcriptase
• restriction endonucelases work by cutting dna at specific sequences of bases called a recognition sequence
• Restriction endonucleases break the DNA molecule by breaking phosphodiester bonds between adjacent nucleotides
• restriction endonucleases cut by either

  • sticky end - overhanging nucleotides
  • blunt end - straight edge
• restriction endonucleases are obtained from bacteria
• reverse transcriptase binds to mRNA and adds complementary nucleotides to the exposed bases joining nucleotides together until a new complementary strand is formed.
• the complementary strand produced from reverse transcriptase is called cDNA = copy DNA
• when a cDNA strand is formed from reverse transcriptase it makes an mRNA cDNA hybrid strand. Then an enzyme destroys the mRNA to leave single stranded cDNA. Then nucleotide bases are added by DNA polymerase to form second strand of DNA leaving us with a DNA fragment.
• PCR = polymerase chain reaction
• PCR takes place in a machine and produces copies of DNA fragments in a continuous cycle
• PCR requires

  • DNA fragment
  • dna nucleotides
  • dna polymerase (taq polymerase)
  • primers
• pcr has three stages:

  1. dna fragment heated to 95 degrees which breaks hydrogen bonds between bases causing strands to separate
  2. temperature cooled to 55 degrees to allow primers to join complementary bases at the end of each strand
  3. Temperature increased to 72 degrees and taq polymerase adds complementary nucleotides to each strand to form two new identical DNA fragments
• Short tandem repeats (STRs) are variable numbers of repetitive non-coding DNA sequences that are directly next to eachother.

  number of repeats varies between different individuals.