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What characteristics make enzymes so useful in industrial processes?
- Specificity: enzymes catalyse reactions between specific chemicals, even in mixtures of many different chemicals. Means fewer by-products are formed and less purification of products is necessary.
- Enzymes can be reused. One enzyme can catalyse reactions repeatedly.
- Temperature: most enzymes function well at relatively low temperatures, much lower than those needed for many industrial chemical processes.
In biotechnological processes previously described, whole organisms are ___ on a ___ __ to generate particular ___. In many areas of industrial processes, product of a single __ __ is needed. It is often more efficient to use ___ ___ to carry out the reaction rather than growing the whole ___ or using an __ ___.
- large scale
- chemical reaction
- isolated enzymes
- isolated enzyme
Isolated enzymes can be produced in large quantities in commercial biotechnological processes. The extraction of enzyme from the fermentation mixture is known as...
Why are immobilised enzymes used in large-scale production?
- Isolated enzymes used can become mixed in with products, and it is costly to seprate the products from the enzymes.
- Therefore, immobilising enzymes means they can continue to catalyse the reaction but do not mix freely with the substrate as they would normally in a cell or isolated system.
Describe the 4 different methods for immobilising enzymes, giving brief advantages and disadvantages for each.
- Absorption: Enzyme molecules mixed with immobilising support and bind to it due to a combination of hydrophobic interactions and ionic links. (absorbing agents include clay, glass beads, resins and porous carbon). Ad: If enzymes held so that active site is not changed and is displayed, then can give very high reaction rates. Disad: Bonding forces aren't particularly strong, so enzymes could become detached (leakage).
- Covalent bonding: Enzyme molecules covalently bonded to an insoluble support (eg. clay), using cross-linking agents. Ad: binding very strong, little leakage. Disad: does not immobilise large quantity of enzymes.
- Entrapment: Enzymes may be trapped, for example in gel bead or network of cellulose fibres. Ad: Trapped in natural state (ie. not bound to other molecules so active site unaffected). Disad: Reaction rates can be reduced because active site is less easily available. Substrates need to get through the trapping barrier.
- Membrane separation: Enzymes held on one side of a partially permeable membrane whilst substrate solution pass along the other side. Ad: active site unaffected, enzymes move freely on one side. Disad: Slower, because of diffusion, can only use small substrates.
Give some general advantages of using immobilised enzymes in large-scale production. (4)
- Enzymes can be reused, so don't have to pay for extra downstream processing costs.
- Allows for quick, continuous process, because enzymes immediately available for reuse.
- No money spent on separating out products and enzymes.
- Immobilised enzymes more stable than free enzymes, because immobilising matrix protects enzyme molecules.
Give some general disadvantages of using immobilised enzymes in large-scale production. (4)
- Rate of enzyme-controlled reactions depend on shape of active site and on the access substrate molecules to the active site. Immobilisation of enzymes can (depending on which one - ones with bonding may affect a.s, but ones with physical separation may affect rate at which subtrate and enzymes collide) affect both of these features.
- Immobilisation requires additional time, equipment and materials.
- Immobilised enzymes can be less active because do not mix freely with substrate. (similar to first point)
- Any contamination costly to deal with - whole system would need to be stopped.
As an intro to gene technologies, list the techniques used in gene technology. (6)
- DNA strands can be cut up into smaller fragmensts using restriction endonuclease enzymes
- Fragments can be separated by size using electrophoresis
- And replicated many times using process called polymerase chain reaction
- DNA fragments can be analysed to give specific base sequence
- DNA fragments can be sealed together using ligase enzymes.
- DNA probes can be used to locate specific sequences on DNA fragments.
a) The sequencing reaction (in later flashcards) can only operate on a length of DNA of about ___ base pairs. b) This means what, when trying to sequence the genome of an organism? c) Also, how do they ensure that assembled code from these fragments is accurate?
- a) 750
- b) Means that genome must be broken up and sequenced in sections.
- c) Sequencing is carried out a number of times on overlapping fragments, with the overlapping regions analysed and put back together to form completed code (using computer program).
Outline the steps involved in sequencing the genome of an organism (don't describe the actual automated sequence process as this will be covered later, but just be sure you understand the link.) [9 steps]
- 1. Genome is cut into smaller fragments (about 100,000 bp) using restriction enzymes.
- 2. Fragments inserted into bacterial artificial chromosomes (BACs) - man made plasmids. Each fragments is inserted into different BAC.
- 3. BACs are then inserted into bacteria (E.coli) - each bacterium contains BAC with different DNA fragment.
- 4. Bacteria divide, creating colonies of cloned cells that all contain a specific DNA fragment. Together the different colonies make a complete genomic DNA library.
- 5. DNA extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNA.
- 6. Fragments separated using electrophoresis.
- 7. Each piece of DNA is sequenced using automated DNA sequencing (described later).
- 8. Computer programmes then compare overlapping regions from the cuts made by different restriction enzymes in order to reassemble the whole BAC segment sequence.
- 9. All the different BAC segment sequences are put together to form the whole sequence of the genome.
Knowing the sequence of bases in a gene of one organism and being able to compare genes for the same (or similar) proteins across a range of organisms is called what?
comparative gene mapping
List the applications of comparing genomes of different species. (5)
- Identification of specific genes for proteins found in all or many living organisms gives clues to relative importance of such genes to life.
- Shows evolutionary relationships.
- Modelling effects of changes to DNA can be carried out for medical research. [Eg. human genes associated with disease like cancer can be found in genomes of mice, and means tests could be done of mice to research disease. Eg2. Test effects of mutations on genes obtained from yeast that are also found in human genome - yeast is haploid, so any mutation to gene is always shown in phenotype.]
- Comparing genomes from pathogenic and similar but non-pathogenic organisms can be used to identify the genes that are most important in causing the disease.
- Understand how genes interact during development (eg. homeobox genes).
List the applications for comparing genomes of the same species. (2)
- Comparisons between genomes of sufferers (of a particular disease) and non-sufferers can b e used to detect particular mutations that could be responsible for the increased risk of disease.
- Develop medical treatments for particular genotypes. In the future, it may be possible to sequence/study a patient's genome so they can receive the most effective medicine.
Only 1.5% of genome of humans actually codes directly for polypeptides/proteins. Much DNA is __-__ ___ and are called __ DNA. However, a lot of them do carry out a number of __ functions (ie influences expression of other genes), many are still yet to be discovered. Therefore ___, the study of whole genomes is important in further understanding roles of genetic information.
- non-coding DNA
DNA is ___ charged because of what?
- negatively charged
- Because of the many phosphoryl (phosphate) groups.
a) Electrophoresis is used to what? b) The process is accurate enough for what? c) What do you need?
- Used to separate DNA fragments based on their size.
- Accurate enough to be able to separate fragments that are different by only one base in length.
- c) Gel 'plate' or slab, containing agarose (sugar), which is covered in buffer solution. Electrodes are attached to each end of gel so that a current can be passed through it.
Outline the basic procedure of electrophoresis.
- 1. DNA samples treated with restriction enzymes to cut them into fragments.
- 2. The DNA samples are placed into wells cut into one end (negative electrode end) of the gel.
- 3. Gel immersed in a tank of buffer solution and an electric current is passed through the solution for a fixed period of time, usually around 2hrs.
- 4. DNA is negatively charged so is attracted to the positive electrode (usually anode), so DNA fragments diffuse through the gel towards +ve electrode end.
- Shorter lengths of DNA move faster than longer lengths and so move further in a fixed time.
- 5. Position of fragments can be shown by using a dye that stains the DNA molecules.
What is the use and basic procedure of Southern blotting?
- Fragments separated in Electrophoresis can be lifted from gel for further analysis. Southern blotting used for this.
- Procedure: Nylon (or other) sheet placed over gel, covered in paper towels, pressed and left over night.
- DNA fragments are now transferred to sheet and can now be analysed.
- DNA fragments are not visible on sheet. So often DNA is labelled with radioactive marker, so that it shows up in photographic film.
What is a DNA probe and what is its use?
A DNA probe is a short single-stranded piece of DNA (around 50-80 nucleotides long) that can be used to identify fragments containing specific sequences of bases. It is complementary to a section of DNA being investigated.
List 2 ways a DNA probe can be labelled.
- Using radioactive marker - location can be revealed by exposure to photographic film.
- Using florescent marker - emits colour on exposure to UV light (used in automated DNA sequencing).
So how do DNA probes work? And what is binding by complementary base pairing known as?
- Copies of the probes can be added to any sample of DNA fragments and, because they are single-stranded, they will bind to any fragment where a complementary base sequence is present. (eg. DNA probes can be incubated with the fragments transfered to nylon membrane after electrophoresis).
- This binding by complementary base pairing is known as annealing.
How are probes useful?
- To locate specific desired gene that is wanted for genetic engineering.
- To identify the same gene on a variety of different genomes, from separate species, when conducting genome comparison studies.
- To identify the presence or absence of an allele for a particular genetic disease (for example, for those who are symptom-less carriers too)
Suggest why it is important that DNA probes are relatively short molecules.
A long probe may partially anneal to a variety of sequences that show some complementary bases. Such as probe may bind in several places and would be of no use in determining the presence of the very specific sequence required.
What does PCR stand for and what is it used for?
- the polymerase chain reaction
- PCR is basically artificial DNA replication. It can be carried out on tiny samples of DNA in order to generate multiple copies of the sample. PCR is repeated over and over to make a lot of copied. The DNA is amplified.
- Esp useful in forensic investigations, for genetic profiling, example.
Outline the stages in PCR.
- 1. DNA sample mixed with supply of free DNA nucleotides and the enzyme DNA polymerase.
- 2. Mixture heated to 95oC, to break the hydrogen bonds holding the DNA double strands together, so making the samples single-stranded.
- 3. Short lengths of single-stranded DNA (around 10-20 bases) that are complementary to the bases at the start of the fragment you want to copy, are added. These are called primers.
- 4. Temp reduced to 55oC, allowing primers to bind (hydrogen bonding) and form small sections of double-stranded DNA at either end of sample.
- 5. Reaction mixture heated to 72oC, which is optimum temp for DNA polymerase. DNA polymerase lines up free DNA nucleotides alongside each template strand. (Like natrual DNA replication). Complementary base pairing means new complementary strands are formed.
- 6. Two copies of the fragment of DNA are formed and one cycle of PCR complete.
- 7. The whole process can be repeated many times so the amount of DNA increases exponentially (x2, x4, x8 etc).
Why are primers needed?
Because DNA polymerase enzymes cannot bind directly to single-stranded DNA fragments.
What is it about PCR that is different to natural DNA replication?
- Can only replicate short sequences of DNA (a few hundred bases long), not entire chromosomes.
- Addition of primer molecules is required in order for the process to start.
- A cycle of heating and cooling is used in PCR to separate and bind strands; DNA helicase enzyme separates strands in natural process.
Ok, so automated DNA sequencing that is needed to sequence whole genomes (talked about before). What two main processes are used here?
- Interrupted PCR
Describe the preparation for the automated DNA sequencing and what is required.
- Reaction mixture (as with PCR) contains many copies of the single-stranded template DNA fragment, free DNA nucleotides, DNA polymerase and primers.
- However, within sequencing mixture, some of the free nucleotides carry a fluorescent marker, and these special nucleotides are modified so that if they are added to the growing chain, the DNA polymerase is 'thrown off' and strand cannot have any further nucleotides added.
- Each nucleotide type has a different coloured fluorescent marker. [ok, now to the procedure...]
Outline the steps involved in automated DNA sequencing. (Remember we already described the preparation mixture for it).
- 1. Primer anneals (joins) at the 3' end (beginning end) of template strand, allowing DNA polymerase to attach.
- 2. DNA polymerase adds free nucleotides, so strand grows (like PCR and natural replication).
- 3. If the special modified nucleotide is added, the polymerase enzyme thrown off and reaction stops on that template strand.
- 4. As reaction proceeds, many molecules of DNA made. The fragments vary in size. In some, the temp strand has only one nucleotide added before polymerase is thrown off by terminator base, in others the template strand is completed. In each case, final added nucleotide is tagged with specific colour (so we know which base it ended with).
- 5. Strands separated like in electrophoresis and ran through machine, which reads the colour sequence, from the strand with only single nucleotide added, to the one with two, three, four and so on. The sequence of colours, and so the sequence of bases, can then be displayed.