Gene technologies 1 Flashcards
(22 cards)
Define genome
The complete set of genes in a cell
Define proteome
The full range of proteins that a cell can produce (coded for by the cell’s DNA / genome)
What is genome sequencing and why is it important?
Identifying the DNA base sequence of an organism’s genome, so amino acid sequences of proteins that derive from an organism’s genetic code can be determined
Explain how determining the genome of a pathogen could allow vaccines to be developed
Could identify the pathogen’s proteome, so could identify potential antigens (proteins that stimulate an immune response) to use in the vaccine
Suggest some other potential applications of genome sequencing projects
- Identification of genes / alleles associated with genetic diseases / cancers. May be used to develop new targeted drugs and gene therapy. Also can screen patients, allowing early prevention / personalised medicine
- Identification of species and evolutionary relationships
Explain why the genome cannot be directly translated into the proteome in complex organisms
- Presence of non-coding DNA
- Presence of regulatory genes
Describe how sequencing methods are changing
- They have become automated (so are faster, more cost-effective and can be done on a larger scale)
- They are continuously updated
What is recombinant DNA technology?
Transfer of DNA fragments from one organism or species, to another
Explain why transferred DNA can be translated within cells of recipient (transgenic) organisms
- Genetic code is universal
- Transcription and translation mechanisms are universal
Describe how DNA fragments can be produced using restriction enzymes
- Restriction enzymes cut DNA at specific base ‘recognition
sequences’ either side of the desired gene - The shape of recognition site is complementary to the active site
- Many will cut in a staggered fashion forming ‘sticky ends’
(single stranded overhang)
Describe how DNA fragments can be produced from mRNA
- Isolate mRNA from a cell that readily synthesises the protein coded for by the desired gene
- Mix mRNA with DNA nucleotides and reverse transcriptase, reverse transcriptase uses mRNA as a template to synthesise a single strand of complementary DNA (cDNA)
- DNA polymerase can form a second strand of DNA using cDNA as a template
Suggest two advantages of obtaining genes from mRNA rather than directly from the DNA removed from cells
- There is much more mRNA in cells making the protein than DNA, so it is easily extracted
- In mRNA, introns have been removed by splicing (in eukaryotes) whereas DNA contains introns, so may be transcribed & translated by prokaryotes who can’t remove introns by splicing
Describe how fragments of DNA can be produced using a gene machine
- Synthesises fragments of DNA quickly and accurately without need for a DNA template. The amino acid sequence of the protein is determined, allowing base sequence to be established
- These do not contain introns so can be transcribed & translated by prokaryotes
Name an in vitro and in vivo technique used to amplify DNA fragments
- In vitro (outside a living organism) - polymerase chain reaction
- In vivo (inside a living organism) - culturing transformed host cells e.g. bacteria
Explain how DNA fragments can be amplified by PCR
- The reaction mixture contains DNA fragment, DNA polymerase, primers and DNA nucleotides
- Heat mixture to 95°C -This separates DNA strands, breaking hydrogen bonds between bases
- Cool mixture to 55°C - This allows primers to bind to DNA fragment
template strand by forming hydrogen bonds between complementary bases - Heat mixture to 72°C - Nucleotides align next to complementary exposed bases, and DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds
Explain the role of primers in PCR
Primers are short, single stranded DNA fragments that are complementary to the DNA base sequence at edges of region to be copied / start of desired gene. They allow DNA polymerase to bind to start synthesis (can only add nucleotides onto pre-existing 3’ end). 2 different primers (forward and reverse) are required (as base sequences at ends are different)
Suggest one reason why DNA replication eventually stops in PCR
There are a limited number of primers and nucleotides which are eventually used up
Summarise the steps involved in amplifying DNA fragments in vivo
- Add promoter and terminator regions to DNA fragments
- Insert DNA fragments and marker genes into vectors (e.g. plasmids) using restriction enzymes and ligases
- Transform host cells (e.g. bacteria) by inserting these vectors
- Detect genetically modified (GM) / transformed cells / organisms by identifying those containing the marker gene (e.g. that codes for a fluorescent protein)
- Culture these transformed host cells, allowing them to divide and form clones
Explain why promoter and terminator regions are added to DNA fragments that are used to genetically modify organisms
Promoter regions allow transcription to start by allowing RNA polymerase to bind to DNA. They can be selected to ensure gene expression happens only in specific cell types.
Terminator regions ensure transcription stops at the end of a gene, by stopping RNA polymerase
What are the role of vectors in recombinant DNA technology?
To transfer DNA into host cells / organisms, usually plasmids or viruses
Explain the role of enzymes in inserting DNA fragments into vectors
- Restriction endonucleases cut vector DNA. The same enzyme is used to cut the gene out so vector DNA & fragments have ‘sticky ends’ that can join by complementary base pairing
- DNA ligase joins DNA fragment to vector DNA, forming phosphodiester bonds between adjacent nucleotides
Explain why marker genes are inserted into vectors
- To allow detection of genetically modified / transgenic cells. If the marker gene codes for antibiotic resistance, cells that survive antibiotic exposure are transformed. If the marker gene codes for fluorescent proteins, cells that fluoresce under UV light are transformed.
- As not all cells will take up the vector and be transformed
