Recombinant DNA Flashcards
What is recombinant DNA technology?
The transfer of fragments of DNA from one organism / species, to another.
Give two reasons why bacteria are able to use human DNA to produce human proteins (2 marks).
- The genetic code is universal
- The mechanism of transcription is universal;
- The mechanism of translation is universal;
List the 5 steps in recombinant DNA technology in the correct order
- Isolation of DNA (usually contains a gene)
- Insertion of DNA into a vector (e.g. a plasmid)
- Transformation of cells (to produce a genetically modfied or transgenic organism with two or more sources of DNA).
- Identification of cells that have taken up the DNA by using marker genes
- Growth / cloning
- i.e. bacterial divide by binary fission
- amplify DNA using PCR
3 ways to isolate a DNA fragment
which typically contains a gene
- Reverse transcriptase & mRNA
- Restriction endonucleases
- Gene machine
Enzyme required to convert
mRNA into cDNA
reverse transcriptase
Describe how isolated mRNA from a cell can be converted into DNA (3 marks).
- mRNA is mixed with free DNA nucleotides AND reverse transcriptase.
- Free DNA nucleotides bind to single stranded mRNA template via complementary base pairing.
- Reverse transcriptase joins DNA nucleotides together to form a single stranded cDNA molecule.
- DNA polymerase is required to make cDNA double stranded.
cDNA means copy DNA i.e. it is a copy based on mRNA
Advantages of using reverse transcriptase to isolate DNA fragments
Introns have been removed
Cells producing protein will contain many mRNA molecules
mRNA is easy to isolate from cells
Disadvantages of using reverse transcriptase to isolate DNA fragments
Many steps involving involving enzyme-controlled reactions
Time consuming
Requires more technical expertise
name of enzyme that isolate a gene / section of DNA from a larger section of DNA e.g. a chromosome.
Restriction endonucleases
Restriction endonucleases are enzymes that [1] phosphodiester bonds at specific DNA base sequences called [2] sites.
hydrolyse
restriction
Restriction sites are often _______________
palindromic
What is the consequence if the restriction site for the restriction endonuclease occurs within the DNA fragment researchers wish to isolate.
This will cut the gene and it will not code for a functional protein.
What type of ends are produced after cutting DNA with restriction endonucleases.
Sticky ends
Blunt ends
What are sticky ends used for?
To insert a gene into a vector
(e.g. a plasmid)
What are blunt ends used for?
Can be amplified by the polymerase chain reaction;
Separated by size using gel electrophoresis;
Advantages of using restriction endonucleases to isolate DNA fragments
- Produce sticky and blunt ends
- 1000s of restriction endonucleases have been isolated that are each highly specific to different DNA sequences
Disadvantages of using restriction endonucleases to isolate DNA fragments
contains introns
Enzymes may cut in the middle of the desired gene leading to a non-functional protein
Which method of DNA isolation is decribed below:
Desired sequence of nucleotide bases are entered into a computer and automated machinery synthesises the DNA fragment
Gene machine
Advantages of using gene machines to isolate DNA fragments
- Faster process owing to automated machinery and fewer enzyme controlled reactions
- Sequences contain no introns
- Blunt and sticky ends can be added
Disdvantages of using gene machines to isolate DNA fragments
If sequence of DNA is unknown, requires the primary structure of the polypeptide to be known.
Once a DNA fragment has been isolated, what must be added to enable gene expression?
A promoter region
(allow transcription factors to bind)
A terminator region
(ensures only the DNA fragment is transcribed)
Suggest and explain one reason why bacteria might not be able to produce every human protein (1 mark).
Cannot splice pre-mRNA, so cannot remove introns
OR
Do not have Golgi apparatus, so cannot process/modify proteins;
OR
Do not have the required transcriptional factors, so cannot carry out transcription/produce mRNA;
A geneticist concluded it would be faster to create a human gene using a gene machine than by using reverse transcriptase to convert mRNA into cDNA.
Suggest why the geneticist reached this conclusion.
faster to use gene machine than all the enzyme-catalysed reactions (involving reverse transcriptase);
Which methods of DNA isolation could used prior to insertion of the human gene into a bacteria to ensure the protein could be produced.
- mRNA & reverse transcriptase AND gene machine
- Produce DNA / human gene without introns;
- Bacteria cannot remove introns / cannot splice mRNA / cannot splice pre-mRNA;
- mRNA & reverse transcriptase AND gene machine
- Produce DNA / human gene without introns;
- Bacteria cannot remove introns / cannot splice mRNA / cannot splice pre-mRNA;
A DNA carrier that can be used to transfer foreign DNA into cells
Examples of vectors
Bacterial plasmid;
Viruses
For insertion to occur, the DNA fragment and plasmid must be cut with the ___________ restriction endonuclease.
same
Describe how enzymes could be used to insert a human gene into a plasmid (2 marks).
- Cut the plasmids with the same restriction endonuclease (used to isolate the gene);
- Both have complementary sticky ends;
- (Mix together) and add DNA ligase to join human gene to the plasmid vector (via phosphodiester bonds);
What bond forms between the sticky ends?
Hydrogen bonds
via complementary base pairing
What bond does DNA ligase form to join the isolated DNA to the plasmid?
Phosphodiester
Describe how a bacterial cell be transformed?
Heat or electric shock;
Creates short-lived pores in the cell surface membrane;
Allows plasmids (with DNA of interest) to be taken up;
Describe binary fission in bacteria
(3 marks).
- Replication of circular DNA;
- Replication of plasmids;
- Division of cytoplasm
(to produce daughter cells);
Why are not all cells transformed?
1) Not all the vectors take up DNA to become recombinant (unsuccessful insertion)
2) Not all the cells take up recombinant vectors
(unsuccessful transformation).
What type of gene is need to identify if a cell has successfully been transformed / is transgenic / genetically modified?
Marker gene
Define a marker gene
allow easy identification of cells that have taken up the vector (e.g. plasmid) with the DNA fragment / gene of interest
3 examples of marker genes
- Green flourescent protein (GFP)
- Antibiotic resistance
- Lactase (which produces a blue product)
What happens if a gene of interest is inserted into the middle of the ampicillin resistance gene in a plasmid?
Damages the gene;
Codes for non-functional protein;
Bacteria will be destroyed if explosed to amplicillin
(no longer resistant to antibiotic);
To enable researchers to identify which bacteria have taken up the plasmid with a human gene, the plasmids contain a gene that codes for a green fluorescent protein (GFP).
Bacteria that contain this plasmid glow green under UV light.
Suggest one advantage of using this gene for GFP to identify bacteria that have taken up plasmids (1 mark).
Can quickly identify transformed bacteria using UV light;
Define in vivo cloning and provide examples
Using a living organism to produce genetically identifcal daugher cells e.g.,
bacteria,
goat,
fish,
crop plant
What does PCR stand for?
polymerase chain reaction
is PCR an example of in vitro or in vivo cloning of DNA
in vitro
The polymerase chain reaction is used to ____________ .
a
amplify DNA
What temperature is required to break the hydrogen bonds in DNA during PCR?
95oC
What is a primer?
Short single stranded DNA molecule;
Has complementary bases to the start of the DNA fragment;
Extended by thermostable DNA polymerase;
What temperature is required for the ‘annealing’* of primers to single stranded DNA?
*annealing is primers binding to each end of the template strands
55oC
What type of DNA polymerase is required for PCR?
thermostable
What temperature is used during the synthesis stage of PCR?
72oC
TRUE or FALSE:
Thermostable DNA polymerases denatures at 72oC?
FALSE
Describe how an isolated gene can be replicated by the polymerase chain reaction (PCR) (4 marks).
- Heat DNA to 95oC to break (weak) hydrogen bonds / separate strands
- Add primers and add DNA nucleotides
- Cool to 55oC to allow ‘annealing’ of primers and DNA nucleotides via complementary base pairing
- Add thermostable DNA polymerase
(e.g. Taq polymerase); - Heat to 72oC
- DNA polymerase joins nucleotides together by phosphodiester bonds to synthesise new strand by extending the primers;
Which formula can be used to calculate the number of DNA strands produced after PCR?
2n
Assuming we started with one DNA molecule, how many DNA molecules are produced after 25 PCR cycles?
2^25 = 3.3x10^7
Assuming we started with 15 DNA molecules, how many DNA molecules are produced after 25 PCR cycles?
15 x 2^25 = 5 x 10^8
How many PCR cycles produced 7230124899 DNA molecules?
log2(7230124899) = 32.8
Why does the number of DNA molecules produced eventually plateau after many PCR cycles?
DNA nucleotides being used up;
so less / nothing to make complementary strands;
OR
Primers used up;
so thermostable DNA POLYMERASE cannot start complementary strands;
Benefits of recombinant DNA technology
Replace faulty or lack of protein
(e.g. insulin in type 1 diabetes)
Gene therapy
(e.g. correct for mutation or lack of gene expression in an organsim)
Improve agriculture
(e.g. larger yields, disease resistance, drought tolerance)
Improve industrial processes
(e.g. produce larger quantities of enzymes more quickly OR improve enzyme-controlled rate of reaction)
Improve understanding of biological processes
(e.g. the regulation of gene expression, DNA replication, mitosis and cell death)
Concerns regarding the widespread use of recombinant DNA technology
- Inserting new genes into a crop plant could disrupt other gene/s function creating toxic products within genetically modified food sources.
- Introducing herbicide resistance genes to crop plants could result in transfer to wild species when they interbreed.
- Technology may become concentrated in the hands of large corporations.
(e.g. technology improves health outcomes but is not accessible to socially and economically disadvantaged groups).
What is gene therapy?
Introduction of healthy gene (which codes for functioning protein) into defective cells (caused by a mutation)
Vectors for gene therapy in humans
Viruses
Liposomes
Describe how a virus acts as a vector in gene therapy.
- Insert isolated DNA fragment or gene of interest into viral genome.
- Virus will insert this gene at the same time as its own genetic material into hosts cells during infection.
- Host cell transcribes and translates gene that codes for functioning protein during replication
Disadvantage of using viral vectors
- May cause an immune response
e.g. formation of cytotoxic T cells / B cells / memory cells
- Not all host cells are successfully infected with the genetically modified virus
Advantage of using viral vectors
- Can enter cells / infect cells / inject DNA into cells;
- Targets specific cells
(attachment proteins bind to receptors); - Replicates in cells
Describe how a liposome acts as a vector in gene therapy.
- Lipid soluble
- So cross the phospholipid bilayer
(and release DNA / gene into cells)
Suggest why the plasmids which contain a gene of interest from a spider - that codes for protein that produce stronger silk fibres - were injected into the eggs of silkworms, rather than into the cells of adult silkworms (2 marks).
- Gene gets into all / most of cells of silkworm;
- So gets into cells that make silk.
The scientists ensured the spider gene - that codes for stronger silk fibre proteins - was expressed only in cells within the silk glands of the silkworm.
What would the scientists have inserted into the plasmid along with the spider gene to ensure that the spider gene was only expressed in the silk glands of the silkworms?
Promoter
the scientists ensured the spider gene - that codes for stronger silk fibre protiens - was expressed only in cells within the silk glands of the silkworm.
Suggest two reasons why it was important that the spider gene was expressed only in the silk glands of the silkworms.
- So that silk fibre protein can be harvested;
- Silk fibre proteins in other cells might cause harm;
