Recombinant DNA technology Flashcards
What is recombinant DNA technology
Transfer of DNA fragments from one organism or species, to another
Why can DNA be translated within cells
Genetic code is universal
Describe how DNA fragments can be produced using restriction enzymes
Restriction enzymes cut DNA at specific base ‘recognition sequences
Many cut in a staggered fashion forming ‘sticky ends’
Describe how DNA fragments can be produced from mRNA
Isolate mRNA from a cell
Mix mRNA with DNA nucleotides and reverse transcriptase
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
Much more mRNA in cells making the protein than DNA
In mRNA, introns have been removed by splicing whereas DNA contains introns
So can 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 & accurately from scratch without need for a DNA template
Determines AA sequence
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 eg. bacteria
Explain how DNA fragments can be amplified by PCR
- Mixture heated to 95
● This separates DNA strands
● Breaking hydrogen bonds between bases - Mixture cooled to 55
● This allows primers to bind to DNA fragment template strand
● By forming hydrogen bonds between complementary bases - Mixture heated to 72
● Nucleotides align next to complementary exposed bases
● DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds
Explain the role of primers in PCR
Primers are short, single stranded DNA fragments
Complementary to DNA base sequence
Allowing DNA polymerase to bind to start synthesis
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
1.Add promoter and terminator regions to DNA fragments
- Insert DNA fragments & marker genes into vectors (eg. plasmids) using restriction enzymes and ligases
- Transform host cells (eg. bacteria) by inserting these vectors
- Detect genetically modified (GM) / transformed cells / by identifying
those containing the marker gene (eg. 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
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
Explain the role of enzymes in inserting DNA fragments into vectors
Restriction endonucleases / enzymes cut vector DNA
Same enzyme used that 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
Describe how host cells are transformed using vectors
Plasmids enter cells
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 cells
If marker gene codes for antibiotic resistance, cells that survive antibiotic exposure = transformed
If marker gene codes for fluorescent proteins, cells that fluoresce under UV light = transformed
As not all cells / organisms will take up the vector
Suggest how recombinant DNA technology can be useful
Medicine
● GM bacteria produce human proteins, gene therapy
Agriculture
● GM crops resistant to herbicides\
Industry
● GM bacteria produce enzymes used in industrial processes and food production
Describe gene therapy
Introduction of new DNA into cells
To overcome effect of faulty cells
Suggest some issues associated with gene therapy
Effect is short lived as modified cells (eg. T cells) have a limited lifespan → requires regular treatment
Immune response against genetically modified cells or viruses due to recognition of antigens
Suggest why humanitarians might support recombinant DNA technology
GM crops increase yields → increased global food production → reduced risk of famine
Gene therapy has potential to cure many genetic disorders
What are DNA probes?
Short, single stranded pieces of DNA With a base sequence complementary to bases on part of a target allele / region
Usually labelled with a fluorescent or radioactive tag for identification
Suggest why DNA probes are longer than just a few bases
A sequence of a few bases would occur at many places throughout the genome
Longer sequences are only likely to occur in target allele
What is DNA hybridisation?
Binding of a single stranded DNA probe to a complementary single strand of DNA
Forming hydrogen bonds / base pairs
Explain how genetic screening can be used to locate specific alleles of genes
- Extract DNA and amplify by PCR
- Cut DNA at specific base sequences using restriction enzymes
- Separate DNA fragments using gel electrophoresis
- Transfer to a nylon membrane and treat to form single strands with exposed bases
- Add labelled DNA probes which bind with target alleles (& wash to remove unbound probe)
- To show bound probe, expose membrane to UV light if a fluorescently labelled probe was used
OR use autoradiography (expose to X-ray film) if a radioactive probe was used
