Chapter 19: Genetic Technology Flashcards
What is genetic engineering?
It is the removal of a gene (or genes) from one organism and the transfer into another so that the gene is expressed in its new host. The DNA that has been altered by this process and which now contains lengths of nucleotides from two different organisms
What is recombinant DNA?
Recombinant DNA is DNA made by joining pieces from two or more different sources.
What is a transgenic organism?
The organism which now expresses the new gene or genes is known as a transgenic organism or a genetically modified organism (GMO).
What is an outline of the steps required to produce a GMO?
1 The gene that is required is identified. It may be cut
from a chromosome, made from mRNA by reverse
transcription or synthesised from nucleotides.
2 Multiple copies of the gene are made using the technique known as the polymerase chain reaction (PCR).
3 The gene is inserted into a vector which delivers the gene to the cells of the organism. Examples of vectors are plasmids, viruses and liposomes.
4 The vector takes the gene into the cells.
5 The cells that have the new gene are identified
and cloned.
What three substances are required for genetic engineering?
Enzymes, vectors and genes coding for easily identifiable substances that can be used as markers.
What is an restriction endonuclease and what does it do?
Restriction endonucleases are a class of enzymes from bacteria which recognise and break down the DNA of invading viruses known as bacteriophages (phages for short). Bacteria make enzymes that cut phage DNA into smaller pieces. These enzymes cut the sugar–phosphate backbone of DNA at specific places within the molecule. This is why they are known as endonucleases. Their role in bacteria is to restrict a viral infection, hence the name restriction endonuclease or restriction enzyme.
How is bacterial DNA protected from the restriction enzymes?
Bacterial DNA is protected from such an attack either by chemical markers or by not having the target sites.
What is the function of a restriction enzyme?
A restriction enzyme binds to a specific target site
on DNA and cuts at that site.
What is the target site?
The target sites, or restriction sites, are specific sequences of bases
How do restriction enzymes cut the DNA?
Restriction enzymes either cut straight across the sugar-phosphate backbone to give blunt ends or they cut in a staggered fashion to give sticky ends.
Sticky ends are short unpaired, staggered ends that
can easily form H bonds with complementary bases
cut using same restriction enzyme.
How can DNA be synthesised from nucleotides?
The sequence of nucleotides is held in a computer that directs the synthesis of short fragments of DNA. The fragments are then joined to make longer nucleotides that can be inserted into plasmids.
What is a plasmid?
They are small, circular pieces of double stranded DNA. They occur naturally in bacteria and often contain genes for antibiotic resistance. They can be exchanged between bacteria(even different species of bacteria).
How are plasmids obtained?
To get the plasmids, the bacteria containing them are
treated with enzymes to break down their cell walls. The ‘naked’ bacteria are then spun at high speed in a centrifuge, so that the relatively large bacterial chromosomes are separated from the much smaller plasmids.
How is a gene inserted into a plasmid vector?
The circular DNA of the plasmid is cut open using a
restriction enzyme. The same enzyme as the one used to cut out the gene should be used, so that the sticky ends are complementary. If a restriction enzyme is used that gives blunt ends, then sticky ends need to be attached to both the gene and the plasmid DNA. The opened plasmids and the lengths of DNA are mixed together. Some of the plasmid sticky ends pair up with the sticky ends on the new gene. The enzyme DNA ligase is used to link together the sugar–phosphate backbones of the DNA molecule and the plasmid, producing a closed circle of double-stranded DNA containing the new gene. This is now recombinant DNA.
What are some properties of a plasmid that make them good vectors? (6)
- Low molecular mass: can be taken up by bacteria easily
- Polylinker: a short length of DNA containing several target sites for different restriction enzymes
- Has one or more marker genes, allowing cells that take up recombinant plasmid to be identified, making it easy to screen.
- An origin of replication so that they can be copied
- Resistant to shearing
- Easy to isolate in large quantities
How are plasmids taken up by bacteria?
The bacteria are treated by putting them into a solution with a high concentration of calcium ions, then cooled and given a heat shock to increase the chances of plasmids passing through the cell surface membrane.
A small proportion of the bacteria, perhaps 1%, take up plasmids with the gene, and are said to be transformed. The rest either take up plasmids that have closed without incorporating a gene or do not take up any plasmids at all.
How can insulin genes be made?
Pancreatic β cells contain large quantities of mRNA for insulin as they are its only source in the body. The mRNA is then incubated with the enzyme reverse transcriptase which comes from the group of viruses called retroviruses. This enzyme reverses transcription, using mRNA as a template to make single stranded DNA. These single-stranded DNA molecules are then converted to double-stranded DNA molecules using DNA polymerase to assemble nucleotides to make the complementary strand.
What is the advantage of make insulin genes through the use of the enzyme reverse transcriptase?
The main advantage of this form of insulin is that there is now a reliable supply available to meet the increasing demand.
What are two examples of genetic markers that can be used?
- Enzymes obtained from jellyfish make a protein called GFP (green fluorescent protein) that fluoresces bright green in ultraviolet light. The gene for the enzyme is inserted into the plasmids. So all that needs to be done to identify the bacteria that have taken up the plasmid is to shine ultraviolet light onto them. The ones that glow green are the genetically modified ones.
- Another marker is the enzyme β-glucuronidase
(known as GUS for short), which originates from E. coli. Any transformed cell that contains this enzyme, when incubated with some specific colourless or non-fluorescent substrates, can transform them into coloured or fluorescent products.
What do promoters do?
The promoter controls the expression of genes. It is the region of DNA to which RNA polymerase binds as it starts transcription.
How does a promoter play a role in gene expression in recombinant plasmid DNA?
If we want the gene that we are going to insert into a
bacterium to be expressed, then we also have to insert an appropriate promoter.
What is gel electrophoresis?
Gel electrophoresis is a technique that is used to separate different molecules. It is used extensively in the analysis of proteins, alleles and DNA. This technique involves placing a mixture of molecules into wells cut into agarose gel and applying an electric field.
What does the movement of charged particles in gel electrophoresis depend on?
■ net (overall) charge – negatively charged molecules move towards the anode (+) and positively charged molecules move towards the cathode (–); highly charged molecules move faster than those with less overall charge
■ size – smaller molecules move through the gel faster than larger molecules
■ composition of the gel – common gels are
polyacrylamide for proteins and agarose for DNA; the size of the ‘pores’ within the gel determines the speed with which proteins and fragments of DNA move.
What happens in the electrophoresis of proteins?
The charge on proteins is dependent on the ionisation of the R groups on the amino acid residues.
Whether these R groups are charged or not depends on the pH. When proteins are separated by electrophoresis, the procedure is carried out at a constant pH using a buffer solution. Usually proteins have a net negative charge.
Gel electrophoresis has been used to separate the
polypeptides produced by different alleles of many genes. For example, allozymes are variant forms of enzymes produced by different alleles of the same gene.
There are also many variants of haemoglobin.
Adult haemoglobin is composed of four polypeptides: 2 α-globins and 2 β-globins. In sickle cell anaemia, a variant of β-globin has an amino acid with a non-polar R group instead of one with an R group that is charged. These two variants of the β-globin can be separated by electrophoresis because they have different net charges. This means that haemoglobin molecules in people who have sickle cell anaemia have a slightly lower negative charge than normal haemoglobin and so the molecules do not move as far through the gel as molecules of normal haemoglobin. The test to find out whether someone carries the sickle cell allele makes use of this difference.
Where do the DNA fragments move in gel electrophoresis?
`To the anode due to carrying a small charge thanks to the negatively charged phosphate groups.
What are VNTRs?
A region of DNA that is known to vary between
different people is chosen. These regions often contain variable numbers of repeated DNA sequences and are known as variable number tandem repeats (VNTRs).
What is genetic profiling?
It is sequencing a length of DNA of one organism and comparing it to another by looking at the ‘variable number tandem repeats’ (VNTRs)
How is gel electrophoresis of DNA carried out?
DNA is extracted from anything that contains cells such as root of hair, blood splatter, saliva and so on. Usually the quantity of DNA is increased by using the polymerase chain reaction (PCR), which makes many copies of the DNA that has been found. The DNA is then chopped into pieces using restriction enzymes known to cleave it close to the VNTR regions. Now the DNA is ready for electrophoresis.
The DNA is placed on agarose gel and current is applied. Fragments travel towards anode, shorter fragments traveling further/faster, than longer ones.
When the current is turned off, the gel contains DNA fragments that have ended up in different places. These fragments are not visible straight away.
To make the fragments visible, they are carefully
transferred onto absorbent paper, which is placed on top of the gel. The paper is then heated just enough to make the two strands in each DNA molecule separate from one another. Short sequences of single-stranded DNA called probes are added; they have base sequences complementary to the VNTR regions. The probes also contain a radioactive phosphorus isotope so when the paper is placed on an X-ray film, the radiation emitted by the probes (which are stuck to the DNA fragments) make the film go dark. So, we end up with a pattern of dark stripes on the film matching the positions that the DNA fragments reached on the agarose gel. Alternatively, the probes may be labelled with a fluorescent stain that shows up when ultraviolet light is shone onto them.
How is the polymerase chain reaction carried out?
- First, the DNA is denatured by heating it to around 95 °C to separate the DNA molecule into its two strands, leaving bases exposed.
- Annealing- A primer(short length of DNA that has a base sequence complementary to the start of the part of the DNA strand that is to be copied) attaches to the start of the DNA strand. This requires a temperature of about 65°C.
- Elongation- Building up complete new DNA strands using DNA polymerase requires a temperature of around 72 °C.
Once the DNA has been copied, the mixture is heated again, which once more separates the two strands in each DNA molecule, leaving them available for copying again. Once more, the primers fix themselves to the start of each strand of unpaired nucleotides, and DNA polymerase makes complementary copies of them.
What does the PCR machine consist of and what does it look like?
The tubes are very small (they hold about 0.05 cm3) and have very thin walls, so when the temperature in
the machine changes, the temperature inside the tubes changes very quickly.
The DNA sample is placed into a tube together with the primers, free nucleotides, a buffer solution and the DNA polymerase. The DNA polymerases used for this process come from microorganisms that have evolved to live in hot environments.
What are some advantages of Taq polymerase?
o It is not easily destroyed by denaturing so doesn’t
have to be replaced every cycle
o High optimum temperature: so temperature for the elongation step does not have to be dropped below that of the annealing process, so efficiency is maximised.
