AQA A2 Biology B21 Recombinant DNA Technology

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C21.1 Producing DNA fragments AQA A2 Biology B21 Recombinant DNA Technology

 

In the following passage replace each number with the most appropriate word or words. Where the DNA of two different organisms is combined, the product is known as recombinant DNA (1) DNA.

 

One method of producing DNA fragments is to make DNA from RNA using an enzyme called reverse transcriptase (2).

 

This enzyme initially forms a single strand of DNA called complementary (3) DNA.

 

To form the other strand requires an enzyme called DNA polymerase (4).

 

Another method of producing DNA fragments is to use enzymes called restriction endonucleases (5), which cut up DNA.

 

Some of these leave fragments with two straight edges, called blunt (6) ends. Others leave ends with uneven edges, called sticky (7) ends.

 

If the sequence of bases on one of these uneven ends is GAATTC, then the sequence on the other end, if read in the same direction, will be CTTAAG (8).

 

21.2 In- Vivo gene cloning – the use of vectors AQA A2 Biology B21 Recombinant DNA Technology : Page No. 275

1

The vector is used to transport the DNA into the host cell. There are different types of vector but the most commonly used is the plasmid. Plasmids are circular lengths of DNA, found in bacteria, which are separate from the main bacterial DNA. Plasmids almost always contain genes for antibiotic resistance, and restriction endonucleases are used at one of these antibiotic-resistance genes to break the plasmid loop.

2

Gene Markers are used to identify host cells. There are a number of different ways of using gene markers to identify whether a gene has been taken up by bacterial cells.

These are needed to identify cells that have successfully taken up a vector and so become transformed. With most of the techniques above less than 1% of the cells actually take up the vector, so a marker is needed to distinguish these cells from all the others. A common marker, used in plasmids, is a gene for resistance to an antibiotic such as tetracycline. Bacterial cells taking up this plasmid are resistant to this antibiotic. So if the cells are grown on a medium containing tetracycline all the normal untransformed cells (99%) will die. Only the 1% transformed cells will survive, and these can then be grown and cloned on another plate.

3

Fluorescent gene markers is a more recent method of finding out whether bacteria have taken up the desired plasmids.

Results can be obtained more easily and more quickly -because, with antibiotic-resistance markers, the bacterial cells with the required gene are killed, so replica plating is necessary to obtain the cells with the gene.

 

With fluorescent gene markers, the bacterial cells are not killed and so there is no need to carry ouc replica plating.

 

4 a

1. C, D. J, K and L -because those that did not take up the plasmid will not have taken up the gene for ampicillin resistance and so will be the ones that arc killed on the ampicillin plate. i.e. the colonies that have disappeared.

 

b

 

E, F and I -because those with the plasmid containing gene X will have a non-functional gene for tetracycline resistance and therefore the colonies will have been killed on the tetracycline plate, i.e. the colonies will have disappeared.

 

 

21.3 In vitro gene cloning – the polymerase chain reaction AQA A2 Biology B21 Recombinant DNA Technology : Page No. 277

 

1 * Primers – short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments * nucleotides – which contain each of the four bases found in DNA * thermocycler – a computer-controlled machine that varies temperatures precisely over a period of time (Figure 1).

2 The primers provide the starting sequences for DNA polymerase lo begin DNA copying because DNA polymerase can only attach nucleotides lo the end of an existing chain.

Primers also prevent the two separate strands front simply rejoining. Synthesis of DNA. The temperature is increased to 72 °C. This is the optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands. It begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain.

3 In a PCR experiment, two primers are designed to match to the segment of DNA you want to copy. Through complementary base pairing, one primer attaches to the top strand at one end of your segment of interest, and the other primer attaches to the bottom strand at the other end.

4 Hydrogen Bond.

5 ]

Because any contaminant DNA will also be multiplied and could lead to a false result.

21.4 Locating genes, genetic screening, and counselling AQA A2 Biology B21 Recombinant DNA Technology : Page No. 285

 

 

]1 ]

A DNA probe is a short, single-stranded length of DNA that has some sort of label attached that makes it easily identifiable. The two most commonly used probes are:

• Radioactively labelled probes, which are made up of nucleotides with the isotope 32P. The probe is identified using an X-ray film that is exposed by radioactivity.

* Fluoroscently labelled probes, which emit light (fluoresce) under certain conditions, for instance when the probe has bound to the target DNA sequence.

2 ]

* A fragment of DNA is produced that has a sequence of bases that are complementary to the mutant allele we are trying to locate.

 

* Multiple copies of our DNA probe are formed using the polymerase chain reaction.

 

* A DNA probe is made by attaching a marker, for example a fluorescent dye, to the DNA fragment.

 

• DNA from the person suspected of having the mutant allele we want to locate is heated to separate its two strands.

 

* The separated strands are cooled in a mixture containing many of our DNA probes.

 

• If the DNA contains the mutant allele, one of our probes is likely to bind lo il because the probe has base sequences that are exactly complementary to those on the mutant allele.

 

* The DNA is washed clean of any unanached probes.

 

• The remaining hybridised DNA will now be fluorescently labelled with the dye attached to the probe.

 

• The dye is detected by shining eight onto the fragments causing the dye to fluoresce which can be seen using a special microscope.

 

3 a ]

 

Tumor suppressor genes can be defined as genes which encode proteins that normally inhibit the formation of tumors. Their normal function is to inhibit cell proliferation, or act as the “brakes” for the cell cycle.

 

Mutations in tumor suppressor genes contribute to the development of cancer by inactivating that inhibitory function. Mutations of this type are termed loss-of-function mutations. As long as the cell contains one functional copy of a given tumor suppressor gene (expressing enough protein to control cell proliferation), that gene can inhibit the formation of tumors.

 

Inactivation of both copies of a tumor suppressor gene is required before their function can be eliminated. Therefore, mutations in tumor suppressor genes are recessive at the level of an individual cell. As we will see, the inactivation of tumor suppressor genes plays a major role in cancer.

 

b ]

He/she might change their lifestyle to reduce the risk of cancer, e.g. by giving up smoking, losing weight, eating more healthily and avoiding mutagens as far as possible; checking more regularly !or early symptoms of cancer; choosing to undergo treatment.

 

21.5 Genetic fingerprinting AQA A2 Biology B21 Recombinant DNA Technology : Page No. 289

 

1 ]
DNA fingerprinting is a method used to identify an individual from a sample of DNA by looking at unique patterns in their DNA.

PCR is an automated procedure that generates lots of copies of a specific sequence of DNA. It only requires small amounts of DNA to start with and can even make copies from a DNA sample that is partially degraded.

In PCR small bits of DNA called primers bind to complementary sequences of the DNA of interest and mark the starting point for the copying of the DNA of interest.

In PCR, the reaction is repeatedly cycled through a series of temperature changes, which allow many copies of the target region to be produced.

PCR has many research and practical applications. It is routinely used in DNA cloning, medical diagnostics, and forensic analysis of DNA.

2 ]

a ]

Suspect B

The genetic imprint obtained from the blood sample from the crime scene corresponds to the genetic imprint of the suspect B

b ]
Genetic fingerprinting can establish whether a person is likely to have been present at the crime scene, although this does not prove they actually carried OUT the crime. Even if there is a close march between a suspect’s DNA and the DNA found at the crime scene, it does not follow that the suspect carried out the crime. Other possible explanations need to be investigated.

Finally, the probability that someone else’s DNA might match that of the suspect has to be calculated. This calculation is based on the assumption that the DNA which produces the banding patterns is randomly distributed in the community. This may not always be the case, for example, it may not apply where religious or ethnic groups tend to have partners from within their own small community.

4 ]

Genetic fingerprints can help in diagnosing diseases such as Huntington’s disease. This is a genetic disorder of the nervous system. It results from a three-base sequence (AGC) at one end of a gene on chromosome 4 being repeated over and over again ~ a son of genetic stutter. People with fewer than 30 repeats are unlikely to get the disease, while those with more than 38 repeats are almost certain to do so. If they have over 50 repeats, the onset of the disease will occur earlier than average.

 

A sample of DNA from a person with the allele for Huntington’s disease can be cut with restriction endonucleases and a DNA Fingerprint prepared. This can then be matched with fingerprints of people with various forms of the disease and those without the disease. In this way, the probability of developing the symptoms, and when, can be determined.

 

Genetic fingerprints are also used to identify the nature of a microbial infection by comparing the fingerprint of the microbe found in patients with that of known pathogens.

 

5

Genetic fingerprinting can be used to prevent inbreeding, which would cause health and reproductive problems. Inbreeding would decrease the gene pool, which could lead to an increase in genetic disorders. Therefore genetic fingerprinting helps identify the least related individuals in a population which can be bred together.

 

Genetic fingerprinting could be used to prove an animal is a pedigree, which would sell for more money. Help identify plants and animals with a desirable allele or gene which can be selected for breeding, which increases the probability of offspring having the characteristic

 

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Practice questions AQA A2 Biology B21 Recombinant DNA Technology : Chapter 21: Page No. 292-293

1 ]

 

(a) ]

(i) ]
1. Has the restriction site (cut by Kpn1);

2. Once;
3. 1000bp from Kpn1 on site of plasmid / ⅓ way along.

(ii) ]
(Most of) plasmid and rest of unknown DNA / rest of recombinant plasmid / rest of plasmid but not 1000 bp part.

(b)

2.

(c) (i) Smaller fragments move further/faster;

Comparing with distance/speed moved by fragments of known size/markers / DNA ladder.

(ii) 1. Large pieces of DNA present;

2. Add up to more than total length of original DNA / plasmid plus inserted DNA;
3. Because this would add undigested to total (original) length.

   2

 

(a) 1. (If injected into egg), gene gets into all/most of cells of silkworm;

2. So gets into cells that make silk;

(b)
The scientists ensured the spider gene was expressed only in cells within the silk glands.

1. Not all eggs will successfully take up the plasmid;
2. Silkworms that have taken up gene will glow;

 

(c)

 

Promoter (region/gene)

(d)
1. So that protein can be harvested;

2. Fibres in other cells might cause harm;

 

3

(a) Restriction / endonuclease;

 

(b) (i)

1. (Acts as a) marker gene;
2. Shows that the (human) gene has been taken up/expressed;
3. (Only) implant cells/embryos that show fluorescence / contain the jellyfish gene.

(ii)
1. Factor IX present in / extracted from milk.

2. Gene only expressed in mammary glands/udder / gene not expressed elsewhere.
3. Do not need to kill sheep (to obtain Factor IX).

 

(c)

(i)
1. Mutation / nucleus/ chromosomes/DNA may be damaged / disrupts genes.

2. May interfere with proteins (produced)/gene expression/ translation.

OR

3. Embryo/antigens foreign.
4. Embryo is rejected/attacked by immune system.

 

(ii)

1. Saves time/money for others.
2. Same work is not repeated / methods can be compared/improved/ amended/ same errors are not made.

 

4

 

(a)

Graph filled at least half the space available.

Axes correct and labelled with titles and units (see table).

Points correct using logs.

Suitable best fit line.

 

(b)

log of the length of this fragment = 3.5;; (depends on the line positioning)

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