DNA Technology Flashcards
Human genome project
- Sequenced the entire human genome
- Can only sequence short fragments at once
- Spilt genome into small sections, sequence, then put back together
- Sequencing methods constantly being updated (chain termination method, high throughput pyrosequencing)
Why is it harder to sequence complex organisms proteomes?
- Many regulatory genes which turn on/off other genes
- Lots of non-coding DNA
- Hard to determine proteins from DNA
Uses of genome/proteome projects
- Understand evolutionary relatedness (phylogeny)
- Monitor pathogens and understand the antigens to develop new vaccines
Gene technology summary
- Isolate target gene using restriction enzymes / gene machine / reverse transcriptase
- Insert gene into vector
- Insert vector into bacteria
- Identify transgenic bacteria using marker gene
- Culture transgenic bacteria
- Extract + purify the protein of the target gene
Isolating a gene using restriction enzymes
- Restriction enzymes cut DNA at specific pallindromic sites called restriction sites
- If there’s a restriction site either side of a target gene, restriction enzymes can cut it out, leaving DNA with unpaired bases/sticky ends
Isolating a gene using reverse transcriptase
- Cells only have two copies of each gene in the nucleus (hard to access)
- Cells that produce protein coded for by target gene contain many mRNA (easy to access)
- Reverse transcriptase enzyme creates complimentary DNA from mRNA strand
Isolating a gene using a gene machine
- Artificially synthesise DNA from scratch
- Joins 25 nucleotides together at once, forming a oligonucleotide
- Join oligonucleotides together to form a synthetic gene
Inserting target genes (in vivo cloning)
- Isolate target gene and add a marker gene, promoter and terminator region
- Isolate vector DNA (plasmid)
- Use same restriction enzyme to cut plasmid so the sticky ends are complimentary to the target gene’s
- Mix vector and target gene with DNA ligase
- DNA ligase reforms phosphodiester bonds, forming recombinant DNA
- Insert vector with recombinant DNA into bacteria, becoming transformed
- Identify transformed bacteria using a marker gene (fluoresce under UV or have antibiotic resistance)
- Select + culture transformed bacteria
Polymerase chain reaction (in vitro cloning)
- Set up reaction mixture containing DNA sample, free DNA nucleotides, primers and DNA polymerase
- Heat mixture to 95°, breaking hydrogen bonds between complimentary base pairs, making DNA single stranded
- Cool to 50°, allowing primers to bind by complimentary base pairing, making DNA double stranded and DNA polymerase to bind
- Heat to 70°, allowing DNA polymerase to join nucleotides, forming phosphodiester bonds
- Repeat, doubling the DNA each cycle
What are primers?
Short sequences of DNA that are complimentary to the start of he DNA sample. Used to select which part of the DNA is sampled
Advantages of GMO’s
Agriculture
- use less chemical pesticide
- more efficient food chain
- preventing diseases from vitamin deficiency
Industry
- fast and cheap
- treat diseases
- pathogens won’t develop resistance
Medicine / pharming
- makes human proteins
- cheaper and easier than synthetic
Disadvantages of GMO’s
Agriculture
- monoculture has low genetic diversity
- have to buy seeds every year
- decrease in biodiversity
Industry
- mutation
- used in war
Medicine / pharming
- unexpected health issues
- animal cruelty
What is gene therapy?
Changing faulty alleles that cause genetic disease. If caused by two recessive alleles, add the dominant allele. If caused by a dominant allele, silence dominant allele so it can’t be transcribed and the recessive allele is expressed
Types of gene therapy
- Germ line gene therapy (changing the alleles of gametes so offspring inherit changes)
- Somatic gene therapy (changing the alleles of body cells)
Problems with gene therapy
- Alleles inserted into wrong locus
- Could silence wrong gene by mistake (tumour suppressor gene)
- Gene could be over expressed
- Could be used for non-medicinal uses
