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Define the term recombinant DNA
DNA that is made by joining pieces from two or more different organisms
State what is meant by Genetic engineering/transfer
The manipulation of genetic material and is usually done to modify a characteristic in the host organism
Outline the stages of Gene transfer
The target gene is identified and may be cut from chromosomes, made from mRNA by reverse transcription or synthesised from nucleotides
Multiple copies of the gene are made using PCR
The gene is inserted into a vector which delivers the gene to the cells of the organisms
The vector takes the gene into the cell
The cells that have the new genes are identified and cloned
What does a genetic engineer need in order to perform gene transfer?
Enzymes - restriction endonucleases, ligase, reverse transcriptase
Vectors - viruses, plasmids
Genes coding for easily identifiable substances that can be used as markers
What is the function of the Polymerase Chain Reaction?
PCR is an in vitro method of cloning and amplifying a fragment of DNA
Outline the substances required for PCR
DNA fragments that’s going to be amplified
Primers (short sections of DNA)
DNA Taq polymerase
Free floating nucleotides
Describe the process of PCR or in vitro
Set up the reaction mixture
Heat the DNA fragments to 95 degrees in order to denature the DNA and break the hydrogen bonds
Cool the mixture at 65 degrees so that the primers can bind with the complementary bases so that a new strand of DNA can be formed (annealing)
Reheat the mixture to 72 degrees so that Taq polymerase can activate and make new complementary strands
Repeat. The number of DNA fragments is doubled each PCR cycle
Why is Taq Polymerase used during PCR?
It is thermophilic so it can withstand high temperatures and doesn’t denature
Why are primers needed for PCR?
They provide a starting point for Taq polymerase and they also prevent the two DNA strands from joining after they’ve been separated
What are recognition sites?
Sections of DNA where the base sequence has antiparallel base pairs
Outline the in vivo process of amplifying DNA fragments
The vector DNA is cut open by restriction endonucleases and now the vector DNA has sticky ends on each side
The DNA fragments with sticky ends that are complementary to the vector are bound to the Vector DNA by DNA ligase
The vector transfers the recombinant DNA to the host cells
Marker genes are inserted so that the transformed cells can be identified if they have recombinant DNA
What is gel electrophoresis?
A technique that is used to separate different molecules and is used a lot in the analysis of proteins and DNA
What is the function of gel electrophoresis?
To separate DNA fragments, nucleic acids and proteins according to their size and charge
State factors that affect the movement of charged molecules within the gel during gel electrophoresis
Net overall charge (negative molecules move to the positive anode)
Size (smaller molecules move faster than larger)
Composition of the gel
Outline the process of Gel electrophoresis
DNA is amplified using PCR and labelled using fluorescent labels
Restriction enzymes cut DNA into fragments and then inserted into a well in the gel
The DNA moves in the gel towards the anode
DNA strands are then separated by heat or alkali
Probes are added to bind to specific DNA sequences and they can show up on X-ray film since they are radioactive
The alleles are then identified
How can different alleles be differentiated during gel electrophoresis?
Different alleles code for slightly different amino acid sequences
The charge of proteins will differ depending on the R group of amino acids present
The proteins can be separated according to the charge
What is a DNA probe?
A short single strand of nucleotides with a label attached. It is complementary to the base sequence of a particular gene in a DNA
The label may be fluorescent or radioactive
What is a plasmid?
A circular DNA structure found in bacteria. It carries DNA into the host cell
Why are plasmids used in genetic engineering?
They are easy to isolate and insert into DNA
They often have a gene for antibiotic resistance which can be used to identify bacteria that have taken up the plasmid
It’s double stranded so it can carry out prokaryotic and eukaryotic DNA
Why do promoters need to be transferred along with the desired gene?
Promoters control the expression of the gene. Transcription factors and RNA polymerase bind to the promoter sequence so transcription can occur
Describe how marker genes are used in gene technology
Marker genes produce a gene product or effect which is easily identifiable so they can confirm that the host cell has taken up the plasmid
What is the role of restriction endonucleases?
They cut viral DNA at specific site
They are part of a bacterium defence against pathogens, but can also be used for gene technology
How is reverse transcriptase used?
It is found in some viruses. It converts mRNA into single-stranded DNA which can produce double-stranded cDNA
Describe how DNA ligase is used in genetic engineering
DNA ligase joins the desired DNA fragment to the opened-up plasmid since they have complementary sticky ends and forms phosphodiester bonds between nucleotides to join the DNA
Outline how microarrays are used to analyse genomes
The DNA from each sample is denatured and then cut into fragments using restriction endonucleases
Each fragments is labelled with a fluorescent tag
The DNA is put onto a microarray that has thousand of probes which hybridise with their complementary genes
UV light is used to see where the DNA has hybridised to the probe and the genomes are identified and compared
How are microarrays used to asses gene expression?
mRNA is extracted from the cell and is converted back into cDNA using reverse transcriptase and DNA polymerase.
The DNA is tagged and hybridised with probes and the fluorescence at each spot shows how much mRNA is there aka the level of expression of gene
What does ‘Bioinformatics’ mean?
The collection, storage and analysis of genomes from thousands of organisms
How is bioinformatics used following genome sequencing?
Once the genomes have been sequences, large databases are created with biological information and this allows for comparisons and analyses to determine similarities and differences between genomes
List uses of bioinformatics
Gene therapy
Evolutionary concepts
Microbial analysis computing
Understanding protein structure
Finding new drugs
In agriculture, to understand crop patterns, pest control and crop management
Give examples of genetic conditions that can be screened for
Sickle-cell
Breast cancer
Haemophilia
Cystic fibrosis
Huntington’s disease
Give benefits of producing human proteins by recombinant DNA technology
The human version of the protein can be produced instead of another animals
The rate at which proteins can be produced is much faster than other methods
Supply is reliable
The gene product is easily extracted and purified
Why i genetic screening carried out?
To test for and diagnose genetic conditions
May be used in personalised medicine to determine the best treatment for a particular genotype
Define the term gene therapy
Treatment of a genetic condition by altering the individual’s DNA
Give examples of vectors that may be used in gene therapy
Viruses
Liposomes
Naked DNA
How can PCR and DNA testing be used in forensic science?
DNA found at the crime scene can be amplified using PCR and tested to see if it matches the DNA of a suspect
Why is genetic engineering used in crops and livestock?
The quality and the yield can be increased by manipulating the genome
Describe how Bt maize is different from regular maize
Bt maise is resistant to certain insects which improves its yield
It expresses the Bt toxin which comes from a species of bacteria. The Bt toxin is selective to certain pests and does not harm other organism
Describe how salmon have been genetically modified
The gene for regulating growth hormone from the salmon and promoter from ocean pout are inserted in a fertilised egg of the Atlantic salmon, the growth hormone is expressed constantly causing the salmon grow faster
How can genetic engineering increase the production of crops?
Crops may be genetically engineered to contain genes for herbicide resistance
Some crops may be engineered to be resistant to insecticides like Bt maize and cotton
Outline the challenges faced when choosing an appropriate vector for gene therapy
Retroviruses insert their DNA randomly into the host cell and this disrupts other genes and could lead to inactivation of tumour suppressor genes
Viral vectors are limited in how much can be carried
Antiviral immunity can prevent viral vectors delivering the DNA to target cells
Naked DNA may not be efficiently delivered into cells
What are the social and ethical considerations of using gene therapy?
Moral acceptability of gene therapy and how it may be exploited
Often expensive so it may only available to the rich
What are the social and ethical considerations of using genetic testing?
Psychological burdens on the induvial and their familied
When screening unborn fetuses, the method of obtaining fetal DNA can cause miscarriage
If the test for genetic condition is positive or if the fetus is not the desired sex, the parents may choose to abort
It can affect insurance covers
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