Recombinant DNA technology
How many fragments would you have after 25 cycles of PCR?
50million
What is a vector?
A vector is a section of DNA that can incorporate another DNA fragment without losing the capacity for self-replication.
The fragments of human DNA and the cut plasmids were mixed together with DNA ligase. Several types of plasmid were formed. Some contained human DNA in the centre of the gene coding for resistance to tetracycline. The different types of plasmid are shown in Figure 2. Explain what causes several types of plasmid to be formed.
All cut DNA have complementary sticky ends. Random process by which sticky ends join
Outline the role of the product of the normal CFTR allele
CFTR is a glycoprotein that transports chloride ions out of cells so that water moves out of cells down its water potential gradient. As a result watery mucus is produced
Some populations of flies are becoming resistant to insecticides intended to kill them. Scientists developed a method for finding out whether a fly was carrying a recessive allele, r, that gives resistance to an insecticide. The dominant allele, R, of this gene does not give resistance. Explain why the scientists used the same restriction endonuclease enzymes on each DNA sample.
Cut DNA at same base sequence to get fragment with gene.
What does CFTR stand for?
Cystic fibrosis transmembrane conductance regulator.
Explain in vivo cloning
A fragment of DNA, containing a single gene or a number of genes, is inserted into a vector that can be amplified within another host cell. , and r. If the fragment of DNA includes one or more genes the process is referred to as gene cloning.
What is a gene probe?
A gene probe is a short strand of DNA (up to 20 bases long) with base sequence that is complementary to part of target gene. Radioactive labelling or fluorescent labelling
Describe the process of PCR in amplifying DNA fragments
A reaction mixture is set up containing the DNA sample, free nucleotides, primers and DNA polymerase. The DNA mixture is heated to 95 degrees celcius to break the hydrogen bonds between the strands of DNA
Replica plating
A simple technique for making an exact copy of an agar plate. A pad of sterile cloth (velveteen pad) the same size as the plate is pressed on the surface of an agar plate with bacteria growing on it. Some cells from each colony will stick to the cloth. If the cloth is then pressed onto a new agar plate, some cells will be deposited and colonies will grow in exactly the same positions on the new plate.The most common use of this technique is in genetic engineering to help distinguish those cells that have taken up a hybrid plasmid vector from those cells that have taken up plasmids without the gene. This is where the second marker gene for resistance to ampicillin is used. If the foreign gene is inserted into the middle of this marker gene, the marker gene is disrupted and won't make its proper gene product. So cells with the hybrid plasmid will be killed by ampicillin, while cells with the normal plasmid will be immune to ampicillin. Since this method of identification involves killing the cells we want, we must first make a master agar plate and then make a replica plate of this to test for ampicillin resistance.
Haemophilia is a genetic condition in which blood fails to clot. Factor IX is a protein used to treat haemophilia. Sheep can be genetically engineered to produce Factor IX in the milk produced by their mammary glands. The diagram shows the stages involved in this process. The jellyfish gene attached to the human Factor IX gene (Stage 2) codes for a protein that glows green under fluorescent light. Explain the purpose of attaching this gene.
Acts as a marker gene. Shows that the human gene has been taken up. Only implant cells that show fluorescence contain the jellyfish gene
The GFP gene can be used as a marker to identify bacteria which have been genetically engineered. Bacteria containing the GFP gene glow green under UV light. Suggest three advantages of using the GFP gene as a genetic marker rather than genes that confer antibiotic resistance.
Antibiotic resistance not transferred. Easier and quicker to identify genetically engineered bacteria. Cheaper because apparatus used isn't as expensive.
The GFP gene can be used as a marker to identify bacteria which have been genetically engineered. Bacteria containing the GFP gene glow green under UV light. Suggest three advantages of using the GFP gene as a genetic marker rather than genes that confer antibiotic resistance.
Antibiotic resistance not transferred; Easier and quicker to identify genetically engineered bacteria. Cheaper because the apparatus used isn't as expensive.
Arabinose is a sugar which binds to the araC protein. The intensity of the green light produced by a bacterium can be changed by varying the amount of arabinose provided to bacteria containing this plasmid. Suggest how arabinose has this effect.
Arabinose reduces inhibition of promoter gene; More arabinose, more mRNA.
When a fragmented DNA is being transferred from the donor to the recipitent, the recipient does not have to be from the same species. Why is this?
Because the genetic code is universal and the transcription and translation mechanisms are also very similar the transferred DNA can be used to produce a protein in the cells of the recipient organism
Not all restriction enzymes produce sticky ends. Some are "blunt cutters," which cut straight down the middle of a target sequence and leave no overhang.Blunt-ended fragments can be joined to each other by DNA ligase. Why then are restriction enzymes that produce sticky ends favored over the enzymes that produce blunt ends?
Blunt ended fragments are harder to ligate together. The ligation reaction is less efficient and more likely to fail because there are no single stranded overhangs to hold the DNA molecules in position.
A husband and wife wanted to know whether they were carriers of the mutated form of a gene. This mutation is a deletion that causes a serious inherited genetic disorder in people who are homozygous. A geneticist took samples of DNA from the husband and the wife. He used a DNA probe to look for the deletion mutation. The DNA probe was specific to a particular base sequence in an exon in the gene. Exons are the coding sequences in a gene. The geneticist compared the couple's DNA with that of a person known not to carry this mutation. The chart shows the geneticist results. The geneticist told the couple they were both carriers of the mutated gene. Explain how he reached this conclusion.
Carriers are heterozygous meaning they have one normal copy and one mutant copy of gene. The couples are carriers because both have DNA that binds to about half the amount of probe that non-carrier does.Probe binds to dominant healthy allele so only one copy of exon in their DNA have the base sequence for probe to bind to.
Explain how heat Shock works to encourage the vector to take up the DNA fragment.
Cells are incubated with the vector in a solution containing calcium ions at 0°C. The temperature is then suddenly raised to about 40°C. This heat shock causes some of the cells to take up the vector, though no one knows why. This works well for bacterial and animal cells.
Explain the limitations of gene therapy as a treatment for cystic fibrosis
Cells have a short life span so repeated treatments required. The target cell uptake of the gene is not 100% successful. Gene insertion may affect the expression of other genes. May produce bad side effects. Repeated treatments may lead to immune response problems.
Genetic engineering could be used to replace the defective gene in either body cells or gametes. At present, gene therapy is limited to replacing genes in body cells.Suggest why replacing genes in gametes is not allowed in the United Kingdom.
Changes to genetic make up of individual may affect normal development.
The scientists wanted to know on which chromosome the gene with alleles R and r was located. From the flies with genotype RR, they obtained cells that were in mitosis and added a labelled DNA probe specific for allele R. They then looked at the cells under an optical microscope. Explain why they used cells that were in mitosis.
Chromosomes would be visible as separate structures and thus the scientists could see to which chromosome the probe was attached.
The scientists wanted to find out whether one of the haplotypes in the Portuguese wolves was the same as one of those in the Spanish wolves. They used a restriction endonuclease, electrophoresis and a labelled DNA probe.Explain why the labelled DNA probe could be used to find out whether the haplotypes were the same.
Complimentary base sequence binds to both haplotypes so label would show up in both.
A bioluminescent jellyfish has a gene which codes for the production of a green fluorescent protein (GFP). Scientists have removed the GFP gene from the DNA of a jellyfish and inserted it into bacteria. Describe how the GFP gene could be removed from the DNA of a jellyfish and inserted into bacteria.
Cut out gene using an endonuclease. The restriction enzyme cuts the base sequence at specific recognition sites called palindromic sequences. When the restriction enzyme makes a cut it leaves sticky ends on the DNA fragment. The same restriction endonuclease is used to cut the plasmid. The DNA fragment is inserted into the plasmid by complemntayr base pairing ligase joins the strands together by forming phosphodiester bonds. Shock treatment is used to help the vector accept the DNA fragment.
A bioluminescent jellyfish has a gene which codes for the production of a green fluorescent protein (GFP). Scientists have removed the GFP gene from the DNA of a jellyfish and inserted it into bacteria. Describe how the GFP gene could be removed from the DNA of a jellyfish and inserted into bacteria.
Cut out gene using restriction endonuclease which cuts the gene at a specific recognition site. When the restriction enzymes cuts it leaves sticky ends. Use restriction enzyme to cut plasmid.Spliced by DNA ligase. Use shock treatment or micropippette to introduce fragment into vector. Transformation.
Cystic fibrosis is a genetic disease caused by an autosomal recessive allele. Gene therapy has been attempted to treat CF since 1993. Outline the basic principles of gene therapy for the treatment of CF.
Cystic fibrosis is caused by a mutation of the CFTR gene. CFTR protein is defective so normal CFTR allele can be inserted into the DNA of the sufferer. A chromosome in cells of respiratory system. The DNA is inserted into a vector called a liopsome and it is taken as spray. Harmless viruses can also be used however not all cells take up virus. The treatment may have unpleasant side-effects. Treatment needs repeating as effects are short lived.
Recombinant DNA
DNA from two different sources that has been joined together
What is DNA ligase and what does it do
DNA ligase is an enzyme that joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA in a process called ligation
Haemophilia is a genetic condition in which blood fails to clot. Factor IX is a protein used to treat haemophilia. Sheep can be genetically engineered to produce Factor IX in the milk produced by their mammary glands. The diagram shows the stages involved in this process. Many attempts to produce transgenic animals have failed. Very few live births result from the many embryos that are implanted. Suggest 2 reasons why very few live births result from the many embryos that are implanted
DNA may be damaged and may interfere with gene expression ( translation and protein production). Embryo foreign so rejected.
A scientist determined the sequences of nucleotides in small samples of DNA obtained from the dried muscle of an extinct mammal. The scientist then compared these DNA sequences with the corresponding DNA sequences in samples obtained from other extinct mammals.The scientist compared the DNA samples obtained from the muscle of one mammal with DNA samples obtained from the bone of another mammal. Explain why this is a valid comparison.
DNA of bone and muscle in the same animal is the same.
The PCR has several stages. The first stage involves a reaction mixture being set up. What does this reaction mixture contain?
DNA sample Free nucleotides Primers DNA polymerase
The mutation responsible for cystic fibrosis is found in a gene coding for a membrane transport protein called CFTR. Patients can receive treatment for cystic fibrosis by inhaling small liposomes containing molecules of DNA with the copy of the normal CFTR gene. Suggest why it was necessary to enclose DNA in liposomes for delivery to cells
DNA will not cross cell surface membrane because it is too large a molecule. DNA is not lipid soluble whilst liposomes are and can therefore fuse with the cell surface membrane.
The scientists added two different primers to each sample of DNA fragments for the polymerase chain reaction. Primer A3 only binds to a 195 base pair fragment from allele r. Primer A4 only binds to a 135 base pair fragment from allele R. Explain why primer A3 and primer A4 only bind to specific DNA fragments
Each has a specific base sequence that is complementary to allele r or R
Explain how the different sized DNA fragments could be separated from each other.
Electrophoresis. Use of electric current and agar gel. Different charged fragments move different distances. Smaller fragments move further.
What is the most efficient method of delivering genes to bacterial cells?
Electroporation. Cells are subjected to a high voltage pulse, which temporarily disrupts the membrane and allows the vector to enter the cell.
A restriction enzyme was used to cut up the human DNA and plasmids. Figure 1 shows the different fragments of human DNA and the type of cut plasmid that was produced. Suggest why the restriction enzyme has cut the human DNA in many places but has cut the plasmid DNA only once.
Enzymes only cut DNA at specific base recognition site. recognition site occurs once in plasmid and many times in human DNA.
What is the difference between exonucleases and endonucleases?
Exonucleases are enzymes that remove nucleotides one at a time from the end of the DNA molecule. Endonucleases are enzymes that hydrolyses bonds within the DNA molecule
True or false? Promoter regions are always present in the vector DNA
FALSE. Promoter regions and terminator regions may be present in the vector DNA or they may have to be added in along with the fragment
Which person, H or G, was heterozygous, Aa? Explain your answer.
G because it only has half the amount of probe for A attaching so it will only produce half the light intensity of H per cycle of PCR.
The diagram shows the base sequence on DNA where a restriction endonuclease cuts DNA. Use evidence from the diagram to explain what is meant by a palindromic recognition sequence on DNA.
GGATCC is the same as CCTAGG in opposite directions
One method of treating people with cystic fibrosis involves the transfer of healthy CFTR genes into epithelial cells in their lungs.Individuals who have been treated by this method do not pass on the healthy CFTR gene to their children. Explain why
Gametes ( reproductive cells) do not contain CFTR allele.
Explain what gene cloning is and why it is important in a range of applications
Gene cloning is the process in which a gene of interest is located and copied out of DNA extracted from an organism.the process of copying fragments of DNA which can then be used for many different purposes, such as creating GM crops, or finding a cure for disease.
What are Type 2 restriction endonucleases used for
Gene cloning. Isolation and Cloning of specific DNA fragments. Restriction mapping.
What are the benefits of using gene technologies?
Gene technologies allow the study and alteration of gene function allowing a better understanding of organism function and the design of new industrial and medical processes.
In genetic engineering, genes from the chromosomes of humans and other organisms can be 'cut out' using enzymes and transferred to cells of other organisms. How else can genes be transffered?
Genes can also be transferred to the cells of animals, plants or microorganisms at an early stage in their development so that they develop with desired characteristics.
What are crop plants that have genes transferred into them called?
Genetically modified crops
Outline the main steps in the PCR that allow many copies of the DNA fragment containng the normal CFTR allele to be produced.
Heat DNA for a short time at 90°C to separate DNA strand. The heat will dentature the DNA by breaking the hydrogen bonds between the complementary base pairs.Cool by lowering the temperature to about 55°C. This allows for the added primers to anneal to the DNA strand. The temperature is raised again to 70°C and Taq polymerase joins the DNA nucleotides to the DNA strand. New strands synthesis and the process repeats itself.
Some people are concerned about using gene therapy to treat genetic disorders in humans. Give 3 possible arguments against the treatment of disorders by gene therapy.
High cost compared with conventional treatments. Adverse effects not known. Other genes introduced which may have damage other genes.
Genetically engineered microorganisms can be used to produce substances that are used in medicine. Name two of these substances.
Hormone e.g. insulin, enzyme; antibiotic, vaccine;
What is a vector containing an additional DNA fragment called?
Hybrid vector
Give three ways in which the information obtained from the use of gene probes might be helpful to a doctor who is counselling someone with a family history of cancer.
Identify carrier of cancer gene. Identify which cancer gene is present. Identify most effective treatment.
What main feature do restriction enzymes have that make them useful for DNA technology?
Importantly, restriction enzymes do not cut randomly; rather, they cut at specific DNA target sequences, which is one of the key features that make them suitable for DNA manipulation
What are two types of gene cloning?
In vivo and using the polymerase chain reaction
Name the 2 mains ways in which DNA fragments can be ammpliefied
In vivo cloning and in vitro cloning
A husband and wife wanted to know whether they were carriers of the mutated form of a gene. This mutation is a deletion that causes a serious inherited genetic disorder in people who are homozygous. A geneticist took samples of DNA from the husband and the wife. He used a DNA probe to look for the deletion mutation. The DNA probe was specific to a particular base sequence in an exon in the gene. Exons are the coding sequences in a gene. The DNA probe the geneticist used was for an exon in the DNA, not an intron. Explain why
Introns are not translated in mRNA. Exons code for amino acids whilst introns do not code for amino acids.Mutations of these exons affect amino acid sequences that produces a change in tertiary structure of protein which makes protein become faulty. So important to know if parents' exons affected rather than any other part of DNA such as introns. Mutations of introns won't affect amino acid sequence.
What does in vivo involve?
Involves the use of restriction enzymes and ligases using vectors and cloning the fragments into host cells.
Name the steps involved in transferring a gene
Isolation Restriction Ligation Transformation Selection Replication
Why do restriction enzymes cut at palindromic sequences?
It is more effective as recognising a palindromic sequence enables them to cut both strands of DNA at the "same" site, because the strand will have the same sequence only in different directions at that site.
Scientists need to take precautions when they carry out restriction mapping. They need to make sure that the enzyme they have used has completely digested the DNA. One check they may carry out is to add the sizes of the fragments together. How could scientists use this information to show that the DNA has not been completely digested? Explain your answer.
Large pieces of DNA present. Add up to more than total length of original DNA plus inserted DNA because this would add undigested to total (original) length
Name the two types of vectors used to deliver the normal functioning CFTR allele to affected cells.
Liposomes and viruses
Explain the methods used for transformation
Marker genes Replica plating
Method of introducing vector
Micropipette, shock treatment, calcium chloride ions, transformation, tungsten bullet.
Why is mRNA used rather than DNA from the target gene?
Most cells only contain 2 copies of each gene making it difficult to obtain a DNA fragment containing a target gene. However theses cells contain many mRNA molecule which are complementary to the gene so mRNA is often easier to obtain
During the PCR process, after denaturation, the temperature is lowered. In theory the 2 strands of DNA could rejoin at this temperature but most of them don't. Explain why.
Most of the hydrogen bonds do not reform because the primer binds to the template DNA strand instead.
What are nucleases?
Nucleases are enzymes that break the bonds linking one nucleotide to the next in a DNA strand.
Genetic engineering could be used to replace the defective gene in either body cells or gametes. At present,gene therapy is limited to replacing genes in body cells. Suggest 3 advantages of replacing a defective gene in a gamete rather than in a body cell
Only has to be treated once. All cells in the body have replaced gene. Gene can be passed to offspring
Transgenic organisms
Organisms that have had DNA from another individual inserted into their genome. Transgenic organisms contain recombinant DNA.
What can PCR be used for?
PCR can be used to make millions of copies of a fragment of DNA in a small period of time.
Describe the role of a genetic counsellor in dealing with genetic diseases in humans and discuss the circumstances in which a couple might be referred to a genetic counsellor.
Pedigree analysis. Genetic screening by using tissue samples from adults. Counscellor will explains results of and dicuss the chances of having affected child. Couple may have to consider termination, alternative treatments and the financial implications of having affected child for the parents and on existing siblings. May discuss ethical issues.A Couple may be referred to a genetic counsecllor if if either of them have, or are carriers of a genetically inherited diseases, the women is older, they ahve ahd misscarriages in the past
Describe how the plasmid is treated in order to form a recombinant plasmid containing the cDNA CFTR gene.
Plasmid is cut using restriction endonucleases which hydrolyses the phosphodiester bond. Enzyme binds to a specific recognition site (palindromic sequence). Restricition enzyme gives plasmid complemnetary sticky ends to that of CFTR gene. Plasmid and CFTR gene sticky ends anneal ( H bonds form) as complementary base pairing occurs. DNA ligase is used to seal up backbone by condensation reaction.
Name the 4 types of vectors
Plasmid vectors Lamda phage vectors Expression vectors Cosmids
Name the reaction used to amplify a small amount of DNA into a larger quantity.
Polymerase Chain Reaction
What are primers?
Primers are short, single stranded pieces of DNA that are complementary to the bases at the start of the fragment you want.
Explain why primers and DNA polymerase would be required in the process of producing a large quantity of DNA.
Primers enables replication and keeps strands apart. DNA polymerase joins nucleotides of new strand together.
The scientists used the polymerase chain reaction (PCR) to produce copies of the cDNA. They added a DNA probe for allele A to the cDNA copies. This DNA probe had a dye attached to it. This dye glows with a green light only when the DNA probe is attached to its target cDNA. Explain why this DNA probe will only detect allele
Probe base sequence complementary to DNA of allele A. Probe binds by forming base pairs so only this DNA has green dye
Describe the steps that are involved in the culture of transformed host cells as an in vivo method to amplify DNA fragments.
Promoter and terminator regions are added to the fragments of DNA if they are not alredy present. Restriction endonucleases and ligases are used to insert fragments of DNA into vectors. Host cells undergo transformation using these vectors. Transformed host cells are identified. Marker genes are used to detect genetically modified (GM) cells or organisms. Transferred host cells are left to grow more producing many copies of the gene
What are promoter and terminator regions?
Promoter regions are DNA sequences that tell the enzyme RNA polymerase when to start transcribing the DNA and produce mRNA. Terminator regions tell it when to stop
What is the importance of having protecting groups?
Protecting groups make sure that the nucleotides are joined at the right points to prevent unwanted branching.
Explain what is meant by recombinant DNA technology
Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another, resulting in translation within the recipient (transgenic organism) due to the universal nature of the genetic code.
The fragments of human DNA and the cut plasmids were mixed together with DNA ligase. Several types of plasmid were formed. Some contained human DNA in the centre of the gene coding for resistance to tetracycline. The plasmids are mixed with the bacteria. Some bacteria take up the plasmids. Explain how it is possible to distinguish between bacteria which have taken up a plasmid with human DNA and those which have taken up a plasmid without any extra DNA.
Replica plating. use of velvet surface to transfer bacteria. use of agar plate containing ampicillin and agar plate containing tetracycline. In bacteria with human DNA tetracycline gene no longer functional so not resistant to tetracycline. Bacteria with human DNA grow on plate with ampicillin but are killed by tetracycline. Bacteria with no extra DNA in plasmid not killed.
What are restriction endonucleases?
Restriction endonucleases are enzymes that break bonds in the sugar phosphate backbones of BOTH strands in a DNA molecule. Each restriction enzyme cuts DNA at a specific recognition sequence.
Explain what is meant by a restriction endonuclease
Restriction endonucleases are enzymes that recognise specific palindromic recognition sequences and cut the DNA at these places
A plasmid can be used to transfer an isolated human gene into a bacterium. Describe how
Restriction endonucleases are used to cut plasmid and sticky ends are produced.DNA ligase is used to fuse phosphodiester bone back together.
Explain how a restriction endonuclease work to leave sticky ends.
Restriction enzymes do not cut directly across the double strand of DNA because this would involve cutting any section of DNA into many different pieces and it would not be easy to remove an entire gene. Instead they cut across the double strands at two different places. The place where they cut across the DNA is called a sticky end. Restriction enzymes can be used to cut out specific genes, and also cut open places in the plasmid DNA where the genes will fit exactly.
Explain the importance of restriction enzymes and sticky ends
Restriction enzymes have an important role in isolating genes, cutting plasmids and producing complementary sticky ends.
Describe two properties that restriction enzymes have that make them useful in DNA technology.
Restriction enzymes have two properties useful in recombinant DNA technology. First, they cut DNA into fragments of a size suitable for cloning. Second, many restriction enzymes make staggered cuts that create single-stranded sticky ends conducive to the formation of recombinant DNA
To make the DNA probe, the geneticist had to find the base sequence of the normal gene. Once he had copies of the gene, what methods would he use to find the base sequence of the gene?
Restriction mapping. Base sequencing of fragments.
Scientists wanted to measure how much mRNA was transcribed from allele A of a gene in a sample of cells. This gene exists in two forms, A and a. The scientists isolated mRNA from the cells. They added an enzyme to mRNA to produce cDNA.Name the type of enzyme used to produce the cDNA.
Reverse transcriptase
mRNA can be converted to cDNA. Name the enzyme used in this process
Reverse transcriptase
What is reverse transcriptase?
Reverse transcriptase is an enzyme that catalyses the process that makes DNA from mRNA
Haemophilia is a genetic condition in which blood fails to clot. Factor IX is a protein used to treat haemophilia. Sheep can be genetically engineered to produce Factor IX in the milk produced by their mammary glands. The diagram shows the stages involved in this process. It is important that scientists still report the results from failed attempts to produce transgenic animals. Explain why.
Saves time and money for others. Same work is not repeated. Methods can be compared and amended. same errors are not made.
What is meant by the term palindromic recognition sequence?
Sequences that consist of antiparallel base pairs
What is a primer?
Short length of single stranded DNA with specific base sequence that indicates where replication starts and stops.
To remove DNA from a cell, the cell membrane needs to be disrupted and the nucleus broken open. Name a detergent that can be used to disrupt the cells.
Sodium dodecyl sulfate is a detergent that can be used to break down cell membranes and cell walls.
State the 3 main processes that occur in the PCR
Strand separation, primer annealing and strand synthesis
Explain the role of Taq polymerase in PCR
Taq polymerase attaches nucleotides to a DNA template, thereby copying the DNA.
Why is Taq polymerase used over DNA polymerase?
Taq polymerase comes from the bacterium Thermus aquaticus, which lives in hot volcanic springs. The enzyme is thermostable and us not denatured by high temparutes
What would happen if the vector didn't contain the promoter region?
The DNA fragment would not be transcribed and so the protein coded for by the DNA fragment will not be produced.
Explain the process of strand separation
The DNA mixture is heated to 95 degrees celsius to break the hydrogen bonds between the strands of DNA. This makes the DNA single stranded and therefore able to act as a template. This is called denaturation
Explain why restriction enzymes have a specific recognition sequence
The active site has a specific tertiary structure which means that only one type of substrate can bind to its active site.
What are the concerns about GM crops?
The concerns include the effect it may have on populations of wild flowers and insects, and uncertainty about the effects of eating GM crops on human health.
What does it mean when a gene is cloned in vitro?
The gene is made in a test tube or in other lab equipment
What's the advantage of using gene machines to make a gene?
The gene it makes doesn't contain introns so the gene can be transcribed and translated in prokaryotic cells
What does it mean when a gene is cloned in vivo?
The gene was made inside a living cell
Explain the method that converts mRNA to cDNA, using reverse transcriptase
The mRNA molecules can be used as templates to make lots of DNA. The enzyme reverse transcriptase makes DNA from RNA template. The DNA produced is called complementary DNA
The scientists used this method with cells from two people, H and G. One person was homozygous, AA, and the other was heterozygous, Aa. The scientists used the PCR and the DNA probe specific for allele A on the cDNA from both people.Explain the curve for person H.
The more probe binding to allele A, the more green light there would be. The light curve goes up exponentially. because the amount of DNA doubles with each PCR cycle.
What is the PCR method use for
The polymerase chain reaction is a method used to create copies of fragments of DNA
Explain how a gene can be created using a 'gene machine'.
The sequence that is required is designed if it doesn't already exist. The first nucleotide in the sequence is fixed to some sort of support for example a bead. Nucleotides are added step by step in the correct order in a cycle of processes that includes adding protecting groups. Short sections of DNA called oligonucleotides are produced. Once these are complete they are broken off from the support and all the protecting groups are removed. The oligonucleotides can then be joined together to make longer DNA fragments
Why do different restriction endonucleases cut at different specific recognition sequences?
The shape of the recognition sequence is complementary to the enzymes active site
Explain the processes of strand synthesis
The temperature is raised again but this time to around 75 degrees celsius, the optimum temperature for Taq polymerase. The Taq polymerase moves along the DNA strand, adding complementary nucleotides by forming phosphodiester bonds.
Explain the process of primer annealing
The temperature is reduced to about 55 degrees celsius so that the primers anneal to the single stranded DNA.
Explain why viruses are good for gene delivery/
The vector is first incorporated into a virus, which is then used to infect cells, carrying the foreign gene along with its own genetic material. Since viruses rely on getting their DNA into host cells for their survival they have evolved many successful methods and therefore an apporpriate choice for gene delivery. The virus must first be genetically engineered to make it safe, so that it can't reproduce itself or make toxins.
Explain the addition of promoter and terminator regions.
The vector must contain specific promoter and terminator regions for the host cell to be able to produce the protein coded for by the DNA fragment
What is the purpose of adding DNA primers?
They bind to the original DNA and signal to the enzyme where to start copying.
In gene therapy, normal genes are put into cells which contain defective genes. In attempts to treat cystic fibrosis, a virus has been used to put the normal gene into cells. Give 3 reasons for using a virus to introduce genes into cells.
They can inject DNA into cells infect cells and replicate in cells.
Name the 3 methods that scientists use to produce fragments of DNA
They use reverse transcriptase to convert mRNA to cDNA. They use a restriction endonuclease to cut DNA fragments out of a large DNA molecule. They uses a 'gene machine' to make the required piece of DNA
What is DNA polymerase?
This is an enzyme that creates new DNA strands
DNA fragments can also be amplified using in vitro cloning. Explain what in vitro cloning is.
This is where copies of the DNA fragments are made outside of a living organism using the polymerase chain reaction.
The genetic code is universal. What does this mean?
This means the same DNA base triplets code for the same amino acids in ALL living things
State the limitations of using liposomes as gene therapy for the treatment of cystic fibrosis.
To maintain the production of the protein, the gene needs to be introduced into the cells lining over and over again because the CFTR gene is not inserted into the genome of the cells. This means that repeated doses of gene therapy are required as the effects are short lived.
What are organisms that contain transferred DNA called?
Transgenic organisms
Naturally occurring restriction endonucleases are categorized into four groups. What are these groups
Type 1 ,2,3, and 4
What's the difference between type 1 and type 2
Type I restriction enzymes cleave DNA randomly away from the recognition site whilst type II restriction enzymes are more useful for laboratory work as they cleave DNA at the site of their recognition sequence.
The fragments of human DNA and the cut plasmids were mixed together with DNA ligase. Several types of plasmid were formed. Describe how the bacteria containing the insulin gene are used to obtain sufficient insulin for commercial use.
Use of fermenters provides nutrients plus suitable conditions for optimum growth. Reproduction of bacteria. Insulin accumulates and is extracted.
The fragments of human DNA and the cut plasmids were mixed together with DNA ligase. Several types of plasmid were formed. Some contained human DNA in the centre of the gene coding for resistance to tetracycline. The plasmids are mixed with the bacteria. Some bacteria take up the plasmids. How is it possible to determine which bacteria have taken up the human insulin gene?
Use of gene probes. Bacteria with insulin gene produce insulin.
At the end of a the ligation reaction three types of cloning vector plasmids will be formed. Name them.
Vector plasmids with the DNA fragment containing the gene required. Vector plasmids with the DNA fragment that doesn't contain the gene required. Vector plasmids with no DNA fragment because the sticky ends have found each other and fused together.
Suggest the features of a virus that would make it a suitable vector for gene therapy for cystic fibrosis.
Virus is able to target epithelial cells. Virus is able to penetrate mucus. Virus is able to get DNA into target cells. Virus is harmless to patient taking it. Virus does not stimulate and immune response.
In gene therapy, normal genes are put into cells which contain defective genes. In attempts to treat cystic fibrosis, a virus has been used to put the normal gene into cells.Give one disadvantage of using a virus to introduce genes into cells.
Virus may cause infection. Virus may be destroyed by immune response or white blood cells.
Research is being carried out into the use of modified viruses to deliver the CFTR gene into cells. Viruses have been shown to be very successful vectors for gene therapy. However, treatments using viruses become less and less successful at delivering the gene into the cells with each repeated treatment. Suggest why
Virus stimulates and immune response. Antibodies are produced as a result of a secondary immune response.
One method of treating people with cystic fibrosis involves the transfer of healthy CFTR genes into epithelial cells in their lungs.Viruses have been used to transfer the healthy CFTR genes into epithelial cells. Give one reason for using a virus to transfer genes into cells.
Virus targets cells and replicates.
Why would it be inappropriate to produce cDNA of the human insulin gene by trying to find mRNA in a small intestine epithelial cell?
Whilst epithelial cells do have the gene to produce the protein insulin their DNA that gene is switched off so insulin is not produced. This means there will be no mRNA for human insulin in the epitheilial cells. Most cells do otr trasncribe all their genes.The cytoplasm of a specialised cell such as epithelial cells only contains mRNA transcribe from some of the genes in its nuclues. As the epithelial cells do not produce the protein insulin because that gene is switched off in epithlaiak cells no mRNA coding for insulin will be found in the cytoplasm of the epitheilial cells. It is rather beta cells in the islets of Langerhans of the pancreas that would have high concentrations of mRNA encoding for insulin
Most restriction enzymes do not cut straight across a DNA molecule instead they separate the strands over a stretch of four bases leaving part of the broken DNA molecule with a short single stranded tail called sticky ends. What is the advantage of sticky ends rather than making clean cuts?
You have more control over which DNA strands join, as a sticky end can only join to a complementary sticky end
What is cDNA
cDNA is the DNA produced from mRNA. Although it is single stranded it can be made double stranded by using DNA polymerase.
Describe a plasmid.
circular DNA separate from main bacterial DNA. Contains only a few genes.
The CFTR gene is found on chromosome 7. The copy of the normal gene can be made using an enzyme called reverse transcriptase. Explain how.
mRNA coding for the CFTR protein is isolated from cells. Reverse transcriptase is used to synthesise a DNA strand that is complementary to the mRNA molecule. The DNA molecule is made double stranded forming cDNA. Sticky ends are added to cDNA. The DNA is inserted into a plasmid to form a recombinant plasmid.
Pancreatic cells produce the protein insulin. They have loads of mRNA molecules complementary to the insulin gene but only 2 copies of the gene itself. Explain how reverse transcriptase can be used to make cDNA from the insulin mRNA
mRNA is first isolated from cells. mRNA is then mixed with free DNA nucleotides and reverse transcriptase. The reverse transcriptase uses mRNA as a template to synthesise a new strand of cDNA.
