Genome projects & gene technologies

• How have sequencing methods improved?

  Were labour-intensive, expensive & could only be done on small-scale. Now automated, cost-effective & done on large-scale. E.g, pyrosequencing.
• What is the genome?

  Entire set of DNA & genes in organism.
• What are genome projects?

  Determine complete genome sequence of an organism. Success depends on complexity of organism & technology available.
• How do gene sequencing methods work?

  Only works on fragments of DNA. To sequence entire genome, chop it up into smaller pieces & sequence them. Pieces put back in order to give sequence of whole genome.
• Sequencing proteomes of simple organisms, e.g, bacteria:

  No introns in DNA- easy to determine proteome from DNA sequence of genome. Useful in medical research & development. E.g, identifying protein antigens on surface of disease-causing bacteria & viruses help develop vaccines. Pathogens can be monitored during outbreaks of disease & antibiotic resistance factors can be identified.
• Sequencing proteomes of complex organisms:

  Contain large sections of non-coding DNA & contain complex regulatory genes- determine when genes that code for a protein switched on or off. More hard to translate genome into proteome.
• What 3 ways can you make DNA fragments?

  Using reverse transcriptase, using restriction endonuclease enzymes & gene machine.
• What are transgenic organisms?

  Organisms that contain transferred DNA. Recipient & donor organisms don't have to be same species.
• What is recombinant DNA technology?


  Combine genetic material from different sources. Transferring fragment of DNA from 1 organism to another. Genetic code universal & transcription + translation mechanisms similar, so transferred DNA can be used to produce protein in cells of recipient organism.
• Making DNA fragments using reverse transcriptase:

  Most cells contain only 2 copies of each gene-hard to obtain DNA fragment with target gene. Cells that produce protein coded for by target gene contain many mRNA molecules complementary to gene-mRNA easier to obtain. Used as templates to make DNA. Reverse transcriptase enzyme makes DNA from RNA template. DNA produced= complementary DNA (cDNA). mRNA isolated from cells & mixed with free DNA nucleotides & reverse transcriptase-uses mRNA as template to synthesis new strands of cDNA.
• Making DNA fragments using a gene machine:


  Sequence required designed. First nucleotide in sequence is fixed to some support, e.g, a bead. Nucleotides added step by step in correct order, in cycle of processes. Includes adding protecting groups-ensure nucleotides are joined at right points, to prevent unwanted branching. Short sections of DNA (oligonucleotides) made. Broken off from support & all protecting groups removed. Oligonucleotides joined to make longer DNA fragments.
• What are sticky ends?

  Small tails of unpaired bases at each end of fragment.
• Making DNA fragments using restriction endonuclease enzymes:

  Some sections of DNA have palindromic sequences of nucleotides. Enzymes recognise specific palindromic sequences (recognition sequences) & cut (digest) DNA at these places. Shape of recognition sequence is complementary to enzyme's active site. If recognition sequences present at either side of fragment wanted, incubate DNA sample with specific enzymes-cuts fragment out via hydrolysis. Cut leaves sticky ends-can be used to anneal fragment to another DNA piece.
• What is in vivo cloning?

  Gene copies made within living organism. As organism grows & divides, it replicates DNA, creating multiple copies of the gene.
• What is in vitro cloning?

  Gene copies made outside of living organism using polymerase chain reaction (PCR).
• What is a vector?

  Used to transfer DNA into cell-can be plasmids or bacteriophages (viruses that infect bacteria).
• What is somatic therapy?

  Altering alleles in body cells, particularly cells most affected by disorder. Doesn't affect sex cells, so any offspring may inherit disease.
• What is germ line therapy?


  Altering alleles in sex cells- so every cell of offspring will be affected by gene therapy & they won't suffer from disease. Illegal in humans.