Chapter 21: Recombinant DNA technology Flashcards
what is meant by recombinant DNA technology?
The transfer of DNA fragments from one organism to another
Why does recombinant DNA technology
work?
- because the genetic code is universal,
- therefore transcription and
translation are also universal mechanisms
therefore all the same DNA base triplets code for same AA in amino
acid sequence across organisms
(indirect evidence of evolution)
what is the name of the organism that receives transferred DNA fragment?
GMO (genetically modified organism) or transgenic
What is recombinant DNA?
- DNA formed artificially by combining DNA from two different organisms
- resulting organism is GMO/transgenic
Briefly describe the stages involved in making a protein using DNA technology (in vivo)
- isolation of DNA fragments (that have gene for desired protein)
NB: Promoter and Terminator must be added! - insertion of DNA fragment into vector
- transformation - transfer of DNA into suitable host cells
- identification of host cells that have successfully been taken up by gene (using gene markers)
- growth/cloning of population of host cells
Name the three processes that can be used in creating DNA fragments
- reverse transcriptase
- restriction endonucleases
- gene machine
Summarise the process of using reverse
transcriptase to produce DNA fragments
- cells that produce protein of interest selected (bc they produce lots of mRNA for that protein)
- mixed w/ free nucleotides which match/bind to complementary base pairs (form H bonds), reverse transcriptase forms sugar-phosphate backbone of cDNA (intron-free)
- to make cDNA double-stranded, add more DNA nucleotides and DNA polymerase
Summarise the process of using restriction endonucleases (enzyme) to produce DNA fragments
- RE complementary to recognition sites/sequences (a DNA base sequence)
- RE cut double stranded DNA to create staggered ends which are palindromic (a staggered cut will create blunt ends)
- sticky ends (exposed bases) aligned next to complementary exposed DNA bases of organism they’re being inserted into - organisms DNA cut with same RE so complementary to each other
(method still contains introns)
Summarise the process of the ‘gene machine’ to produce DNA fragments
- examine protein of interest and find AA sequence
- then mRNA sequence – then DNA sequence
- DNA base sequence entered into computer and oligonucleotides produces
- oligonucleotides = small sections of overlapping single strands of nucleotides that make up gene – joined to create DNA for entire gene
- PCR used to amplify and make double strand, using sticky ends the gene can be inserted into plasmid DNA
In which two ways can we amplify DNA
fragments?
● In vitro / polymerase chain reaction (PCR)
● In vivo / using host cells
Describe the reaction mixture in the first stage of PCR
vessel in thermocycler of PCR
contains:
- the DNA fragment to be amplified
- primers that are complementary to the start of the fragment
- free nucleotides to match up to exposed bases
- DNA polymerase to create the new DNA
Summarise the process of amplifying
DNA fragments using PCR
- Heat DNA to break H bonds between bases, separates DNA strands (95°C)
- Cooled to allow primers to bind (55°C), H bonds form to hold primers in place, nucleotides attach; by complementary base pairing;
- Heated again for optimum temp for DNA polymerase tojoin nucleotides together (72°C)
- New DNA acts as template for next cycle
what is a DNA primer? And the role of a DNA primer?
primer = short, single-stranded DNA sequence
- provides starting sequences for DNA polymerases as they can only attach new DNA nucleotides to an existing strand of nucleotides
Name the three stages of PCR
- Deanturing (95°C)
- Annealing (55°C)
- Synthesis (72°C)
Summarise the process of inserting a
DNA fragment into a vector
- plasmid is used as the vector, and is cut using the same RE used to cut out DNA fragment (so ends are complementary)
- DNA ligase joins the fragment and plasmid together
state the role of DNA ligase
catalyses the (condensation) reaction between nucleotides to form phosphodiester bonds
summarise the process of inserting vector into host cell (i.e. cell transformation)
- vectors/host cells mixed in ice-cold solution, then ‘heat-shocked’ (large,rapid change in °C)
- mixed with Ca2+ ions which also increase permeability of cell membrane of host cell (for vector to be taken up)
state three issues that occur that prevent cell transformation
- recombinant plasmid doesn’t get inside host cell (despite increased permeability of cell membrane )
- plasmid re-joins before DNA fragment entered
- DNA fragment sticks to itself (creating small loop), instead of inserting into plasmid
why do promoters and terminators need to be added to extracted DNA fragment at the start?
promoter = binding site for RNA polymerase to enable transcription to occur terminator = end of gene, causes RNA polymerase to detach and stop transcription SO only one gene at a time is copied into mRNA
Summarise the process of identifying
transformed cells: Genes
marker genes e.g. gene coding for fluorescence can be inserted into vectors along with DNA
when cells grow, UV light can be used to identify which cells have taken up vector and which haven’t
what are marker genes?
- genes that can be used to identify which bacteria successfully took up the recombinant plasmid
- added to vector at same time DNA fragment is
e.g. Antibiotic resistance
Fluorescence
Enzyme markers
Summarise the process of identifying
transformed cells: fluorescent markers
- gene with coding for fluorescence can be inserted into vectors along with the DNA - when cells begin to grow, UV light can be used to identify which cells have taken up the vector and which haven’t
or DNA fragment is inserted into gene that codes for fluorescence, it disrupts gene, so when grown and UV light used, the transformed cell can be identified as the one that does not glow
If a gene for antibiotic resistance is disrupted, what will happen when the bacteria is incubated with that antibiotic?
It will not grow on that antibiotic
i.e. it will die/be killed
If a marker gene for antibiotic resistance is added and the cell is transformed, what will happen when the bacteria is incubated with that antibiotic ?
It will grow on that antibiotic
i.e. not be killed by antibiotic
Summarise the process of identifying
transformed cells: enzyme markers
- e.g. lactase turns substances colourless to blue
- gene for this enzyme inserted into plasmid, DNA fragment inserted into middle of this gene, disrupting it
- bacteria grown in agar plate with colourless substance
- colonies which can’t turn from colourless to blue contain recombinant plasmid
What is the next step after successfully transformed cells have been identified?
- fermenter used to grow multiple copies of host cell with recombinant plasmid
- large, cloned population of host cell can then produce the protein coded for by inserted DNA fragment
What must the vector contain for the host cells to produce the protein coded by the DNA fragment?
Promoter and terminator regions
NB: These may already be in the vector DNA
State the advantages and disadvantages of using reverse transcriptase to extract DNA fragment
advantages:
lots of mRNA (from cell that produces proteins)
disadvantages:
more time-consuming and difficult (i.e. there are more steps)
State the advantages and disadvantages of using restriction endonucleases to extract DNA fragment
advantage: ‘sticky ends’ on DNA fragment make it easier to insert to make recombinant DNA
disadvantage: still contains introns (requires extra step to remove them)
State the advantages and disadvantages of using the gene machine to create DNA fragment
advantages:
- any sequence of nucleotides can be produced, in short time and with accuracy
- artificial genes intron-free or other non-coding regions of DNA so can be transcribed and translated by prokaryotic cells
disadvantage:
- need to know the sequence of amino acids or bases
advantages of PCR
- rapid
- doesn’t require living cells
- automated: more efficient
- only replicates DNA of interest
disadvantages of PCR
- risk of contamination - multiple copies made
advantages of in vivo cloning
- involves almost no risk of contamination (because of complementary stick ends, contained DNA wouldn’t be taken up by plasmid)
- very accurate (contrast to in vitro where contaminant DNA will be copied in subsequent cycles)
- cuts out specific genes
- useful when introducing gene into another organism (as involves use of vectors, plasmid deliver gene to another organism - gene therapy)
- relatively cheap method
disadvantages of in vivo cloning
- slow process as some bacteria grow slowly
Explain why two different primers are required in in vitro gene cloning
because sequences at opposite ends of the two strands of DNA are different
What are the advantages of recombinant technology in agriculture?
- Increase the nutrition of food
- Plants resistant to pests/droughts
- Lowers costs /less environmental problems as less pesticides needed
What are the advantages of recombinant DNA technology in industry?
- Enzymes produced which speed up processes
- More product for less money
What are the advantages of recombinant DNA technology in medicine?
Drugs and vaccines made quickly/cheaply
More affordable and available
What are the disadvantages of recombinant DNA technology in agriculture?
- Monoculture reduces biodiversity
- Superweeds (plants that have developed resistance to one or more herbicides)
- Organic farmers’ crops contaminated
- High prices of crops which need repurchasing each year
What are the disadvantages of recombinant DNA technology in industry?
- Introduction of toxins into food industry
- Large biotech companies outcompete smaller businesses
What are the disadvantages of recombinant DNA technology in medicine?
Limit use of life-saving technologies
Designer babies
What is gene therapy?
Altering defective genes (mutated alles) to treat genetic disorders and cancer
what is a DNA probe? Describe the two common types.
short, single-stranded DNA length with label attached that makes it identifiable
common probes:
1. radioactively labelled probes
made up of nucleotides and isotope, identified using x-ray film exposed radioactively
2. fluorescently labelled probes
emit light under certain conditions e.g. when probe has bound to target DNA sequence
Descibe how a DNA probe works
- probe designed so sequence is complementary to DNA sequence being tested for (i.e. mutant allele)
- amplify DNA base sequence for probe by PCR
- individual’s DNA extracted and heated to separate strands, probes added (mixed in)
- complementary bases from DNA probe bind to DNA sequence - DNA HYBRIDISATION (form H bonds)
- DNA washed clean of unbound probes, bound probes emit light (under UV light)
what is DNA hybridisation?
- the process of combining two complementary single-stranded DNA (allows them to form H bonds between complementary bases so become double-stranded)
What does DNA hybridisation allow for?
- degree of differences between 2 strands of DNA to be studied
- can be used to compare someone’s DNA to a certain gene/allele to see if they have it
Give some applications of DNA probes
● To screen someone’s DNA for a particular heritable health condition. ● To identify a gene for use in genetic engineering. ● To predict how someone will respond to a drug
where can base sequences for mutant alleles be found?
genetic libraries
why must donor DNA be single-stranded when testing for mutant allele using DNA probe?
- to allow for DNA hybridisation between DNA probe and Donor’s DNA
- depends on the formation of H bonds between complementary base sequences
what are the benefits of genetic counselling/profiling?
- identify heritable diseases very early
- can adjust lifestyle to prevent disease/reduce its impact
- treatment can be personalised
what are the disadvantages of genetic counselling/profiling?
- increases stress/anxiety
- results may be inconclusive/uncertain
describe what is meant by ‘personalised medicine’
- an advantage of genetic screening
- doctors can determine how an individual will react to a drug and dosage which’ll produce desired outcome
- saves money (not over-prescribing drugs)
- also helps avoid medicine which could CAUSE HARM
what is genetic fingerprinting?
diagnostic tool involving examination of VNTRs and can be used to determine variability within a population
Describe the principle of genetic fingerprinting
- examines VNTRS (non-coding regions of DNA)
- more closely related = more similar VNTRs
- probability two individuals have the same VNTRs is very low
what does VNTRs stand for?
Variable Number Tandem Repeats
briefly summarise the process of genetic fingerprinting
- DNA sample obtained, VNTRs cut out
using restriction enzymes, labelled, and
cloned using PCR. - Fragments separated using gel electrophoresis.
- Banding patterns of each sample can then be compared
how does gel electrophoresis work?
- DNA fragment placed at one end of agar gel
- Electrical current applied, causing DNA fragments to move towards other end of gel
- shorter fragments travel further
- pattern of bands created is unique to every individual (except identical twins)
Name the five steps involved in genetic fingerprinting
- Extraction
- Digestion
- Separation
- Hybridisation
- Development
explain the process of genetic fingerprinting
- DNA extracted and amplififed by PCR e.g. hair follice
- RE cuts DNA (VNTRs) into fragments (RE complementary to recognition sites before and after VNTRs so length of VNTRs maintained)
- DNA samples separated by gel electrophoresis (DNA has -ve charge so moves to +ve end of agar plate, gel creates resistance, smaller fragments move faster and further)
- DNA made single-stranded (by immersing gel in alkaline solution) and DNA probes complementary to DNA base sequence of VNTRs added, rinse to remove unbound probes
- VNTRS and probes transferred to nylon sheet, where they’re exposed to x-rays to visualise position of radioactive gene probes (UV light is fluorescent probes used)
What is the difference between a DNA probe or a primer?
DNA probe: identifiable label, used in DNA hybridisation, stand alone unit
Primer: no label, occurs in in vitro gene cloning
why don’t inheritance of VNTRs influence on phenotype?
because non-coding regions of DNA
what do points in gel electrophoresis correspond to?
position of DNA fragments as separated during electrophoresis
Give applications of genetic fingerprinting
● Forensics/Crime scenes e.g. to identify victims or suspects.
● Medical diagnosis e.g. to identify type of haemoglobin produced by an individual to diagnose sickle cell anaemia.
● Animal and plant breeding e.g. breed out harmful alleles, ensure pedigree (make sure two organisms aren’t too closely related before selectively breeding them)
describe ways an individual could modify their lifestyle if genetic profiling reveals they are at risk of a disease
- quit smoking
- losing weight
- eating more healthy
- avoiding mutagens (e.g. radiation)
give two ways in which PCR differs from transcription
- transcription uses RNA nucleotides, PCR uses DNA nucleotide bases (uracil v thymine)
- transcription involves stop/start codons (PCR just uses primers)
explain why base pairs are a suitable way of measuring the length of a piece of DNA [2 marks]
- DNA made up of base pairs
- Each base pair is same length
Explain one way in which the PCR differs from DNA replication in a cell [2 Marks]
- primers added in PCR
- to initiate replication
OR - PCR replicates pieces of DNA
- because DNA has been cut
OR - uses heat
- to separate strands
Explain why it takes longer to obtain a genetic fingerprint if the sample is:
- very small [1 mark]
- contaminates [2 marks]
- PCR needed
- other DNA present
need to identify ‘required’ DNA from the rest
why are DNA primers added during PCR? [1 Mark]
- to make beginning/end of part of DNA needed
what is meant by gene therapy?
introduction of a healthy gene/’replacement’ of defective gene
