8.3 and 8.4 Flashcards
Define genome and proteome:
Genome- complete set of genes in a cell
Proteome- full range of proteins that a cell can produce
What is genome sequencing and why is it important?
Identifying the DNA sequence of an organisms genome
So amino acid sequences of proteins that derive from an organism’s genetic code can be determined
Explain how determining the genetic code of a pathogen could allow vaccines to be develeoped:
Could identify the pathogens proteome
So could identify potential antigens to use in the vaccine
Suggest some other potential applications of genome sequencing projects:
Identification of genes/alleles associated with genetic diseases/cancers
-new targeted drugs/gene therapy can be developed
-can screen patients allowing early prevention/personalised medicine
Identification of species and evolutionary relationships
Describe how sequencing methods are changing:
They have become automated
They are continuously updated
What is recombinant gene 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
-Shape of recognition site complementary to active site
Many cut in a staggered fashion forming sticky ends
Describe how DNA fragments can be produced from mRNA:
Isolate mRNA from a cell that readily synthesises the protein coded form 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
DNA polymerase can form a second strand of DN using cDNA as template
Suggest the advantages of obtaining genes from mRNA rather than directly from the DNA removed from cells:
Much more mRNA in cells making the protein that DNA so easily extracted
In mRNA, introns have been removed by splicing whereas DNA contains introns
-so can be transcribed and translated by eukaryotes who can’t splice
Describe how fragments of DNA can be produced using a gene machine:
Synthesises fragments of DNA quickly and accurately from scratch without need for a DNA template
- Amino acid sequence of protein determined, allowing base sequence to be established
These do not contain introns so can be transcribed and translated by prokaryotes
Name an in vivo and an in vitro technique used to amplify DNA fragments:
In vitro- polymerase chain reaction
In vivo- culturing transformed host cells e.g. bacteria
Explain how DNA fragments can be amplified by PCR:
Mixture: DNA fragment, DNA polymerase, primers and DNA nucleotides
- Mixture heated to 95ºC
- separates DNA strands, breaking H bonds between bases - Mixture cooled to 55ºC
- allows primers to bind to DNA fragment template strand, by forming H bonds between complementary bases - Mixture heated to 72ºC
- nucleotides align next to complementary exposed bases
-DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds
Cycle repeated- in every cycle, the amount of DNA doubles causing an exponential increase
Explain the role of primers in PCR:
Primers are short, single stranded DNA fragments
Complementary to DNA base sequence at edges of region to be copied/start of desired gene
Allowing DNA polymerase to bind to start synthesis
Two different primers are required, as base sequence at ends are different
Suggest one reason why DNA replication in PCR usually stops:
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 using restriction enzymes and ligases
Transform host cells by inserting these vectors
Detect genetically modified/transformed cells/organisms by identifying those containing the marker gene
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: allows transcription to start by allowing RNA polymerase to bind to DNA
Can be selected to ensure gene expression happens only in specific cell types
Terminator: Ensure transcription stops at 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
Explain the role of enzymes in inserting DNA fragment fragments into vectors:
Restriction endonucleases cut vector DNA
- same enzyme used that cut the gene out so vector DNA and fragments have same sticky ends that can join by complementary base pairing
DNA ligase joins DNA fragment to vector DNA
- forms phosphodiester bonds
Describe how host cells are transformed using vectors:
Plasmids enter cells (using heat shock in a calcium ion solution)
Viruses inject their DNA into cells which is then integrated into host DNA
Explain why marker genes are inserted into vectors:
To allow detection of genetically modified/transgenic cells/organisms
- IF marker gene codes for antibiotic resistance, cells that survive antibiotic exposure= transformed
- If marker genes code for fluorescent proteins, cells that fluoresce under UV Light=transformed
As not all cells will take up the vector and be transformed
Suggest how recombinant DNA technology can be useful:
Medicine:
-GM bacteria produce human proteins so more ethically acceptable that using animal proteins and less likely to cause allergic reactions
-GM animals/plants produce pharmaceuticals - cheaper
-Gene therapy
Agriculture:
-GM crops resistant to herbicides and insect attack
-GM crops with added nutritional value
-GM animals with increased growth hormone production
Industry:
-GM bacteria produce enzymes used in industrial processes and food production
Describe gene therapy:
Introduction of new DNA into cells, often containing healthy/functional alleles
To overcome effect of faulty/non-functional alleles in people with genetic disorders
Suggest some issues associated with gene therapy:
Effect is short lived as modified cells have a limited life span so requires regular treatment
Immune response against genetically modified cells or viruses
Long term effect not known- could be unknown side effects
