8.2 recombinant DNA technology

Question 1

what is recombinant DNA?

Answer 1

the DNA of two different organisms that has been combined by isolating the desired genes, cloning them and transferring them into microorganisms.

Question 2

organisms which use recombinant DNA are know as what organisms?

Answer 2

transgenic or genetically modified organisms (GMO)

Question 3

explain how transgenic organisms can produce proteins using recombinant DNA.

Answer 3

• the production of proteins in organisms is universal, meaning that the mechanisms of transcription and translation are essentially the same in all organisms.
• transferred DNA can be transcribed and translated within the cells of the transgenic organism, and the proteins it codes for can be manufactured in the same way as they would be within the donor organism.

Question 4

give the five stages involved in the process of producing a protein using the DNA technology of gene transfer.

Answer 4

• isolation of the DNA fragments that contain the gene for the desired protein.
• insertion of the DNA fragment into a vector.
• the transfer of DNA into suitable host cells.
• identification of the host cells that have been successfully taken up by the gene, by use of gene markers.
• growth / cloning of the population of host cells.

Question 5

what is a vector?

Answer 5

a carrying unit used to transport the desired fragment of DNA into the host cell.

Question 6

before a gene can be transplanted, it must be identified and isolated from the rest of the DNA. give three methods which can be used to produce DNA fragments.

Answer 6

• conversion of mRNA to cDNA (complementary DNA) using reverse transcriptase.
• use of restriction endonucleases to cut fragments containing the desired gene from DNA.
• manufacturing the gene in a gene machine.

Question 7

reverse transcriptase is an enzyme. what is the role of reverse transcriptase?

Answer 7

reverse transcriptase catalyses the production of DNA from RNA.

Question 8

explain how reverse transcriptase can be used to produce cDNA from mRNA.

Answer 8

• a cell that readily produces the protein is selected.
• these cells have large quantities of the relevant mRNA, which is therefore more easily extracted.
• the cDNA is made up of nucleotides that are complementary to the mRNA.
• to produce the second strand of DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides on the cDNA template. this double strand of DNA is the desired gene.

Question 9

what are restriction endonucleases?

Answer 9

enzymes which cut a DNA double strand at a specific sequence of bases, known as a recognition sequence.

Question 10

explain why different restriction endonucleases have different restriction sites.

Answer 10

• restriction endonucleases, like all enzymes, have a specific active site complementary in shape and charge distribution to their substrate.
• the specific sequence of nucleotides at the restriction site give the site a highly specific shape and charge distribution, as the sequence of nucleotides are different for different enzymes.
• this means that a large range of restriction endonucleases, each with its own specific restriction site, is possible.

Question 11

give the name of the ends that result from a cut between two opposite base pairs by restriction endonuclease.

Answer 11

blunt ends.

Question 12

explain how restriction endonucleases cut DNA to result in ‘sticky ends’.

Answer 12

• some restriction endonucleases cut DNA in a staggered manner.
• this leaves an uneven cut in which each strand of the DNA has exposed, unpaired bases.
• the two sequences are palindrome (opposites of each other)
• when restriction endonucleases cut DNA in this way it results in sticky ends.

Question 13

it is now possible to manufacture genes in a laboratory using a ‘gene machine’. explain how the amino acid sequence of the desired protein is determined, and how the complementary DNA triplets are worked out.

Answer 13

• the desired sequence of nucleotide bases of a gene is determined from the desired protein.
• this allows the amino acid sequence of this protein to be established.
• from this, the mRNA codons are looked up on a database, and the complementary DNA triplets are worked out.

Question 14

the desired sequence of nucleotide bases for the gene is fed into a computer. explain the process by which the computer assembles this nucleotide sequence into the desired gene.

Answer 14

• the computer designs a series of small, overlapping single strands of nucleotides, called oligonucleotides.
• these oligonucleotides are then joined together to construct the desired double-stranded gene.

Question 15

through which process is the desired gene replicated?

Answer 15

the desired gene is replicated using the polymerase chain reaction.

Question 16

using sticky ends, the gene can then be inserted into a bacterial plasmid. what does this bacterial plasmid act as?

Answer 16

the bacterial plasmid acts as a vector for the gene, allowing it to be stored, cloned or transferred to another organism in the future.

Question 17

give two advantages of using a gene machine to manufacture a desired gene.

Answer 17

• any sequence of nucleotides can be produced in a very short time.
• the artificial genes manufactured by the gene machine are free of introns and other non-coding DNA, so can be transcribed and translated by prokaryotic cells.

Question 18

for the transcription of any gene to take place RNA polymerase must attach to the DNA near the gene. where does RNA polymerase bind to?

Answer 18

the binding site for RNA polymerase is a region of DNA known as a promoter region.

Question 19

give the two ways in which genes can be cloned so that there is a sufficient quality for medical or commercial use.

Answer 19

• in vivo, by transferring the fragments to a host cell using a vector.
• in vitro, using the polymerase chain reaction.

Question 20

what is ‘in vivo’ cloning?

Answer 20

the cloning of genes within living organisms.

Question 21

which enzyme is used to combine two sections of DNA from different sources to produce recombinant DNA?

Answer 21

DNA ligase.

Question 22

how does DNA ligase combine two sections of DNA.

Answer 22

once the complementary bases of the two sticky ends have paired up, DNA ligase is used to bind the phosphate-sugar backdown of the two sections of DNA, to form one molecule of recombinant DNA.

Question 23

what is a terminator region?

Answer 23

a terminator region releases RNA polymerase to end transcription.

Question 24

explain how a fragment of DNA is inserted into a bacterial plasmid.

Answer 24

• the same restriction endonucleases that were used to cut the DNA fragment is used to break the plasmid loop.
• the sticky ends on the opened up plasmid are complementary to the sticky ends on the DNA fragment.
• the DNA fragments become incorporated with the opened up plasmid, and they are joined by DNA ligase. the plasmids now contain recombinant DNA.

Question 25

once the DNA fragments have been incorporated into at least some of the plasmids, they must then be reintroduced into bacterial cells. through which process is this achieved?

Answer 25

this process is called transformation, and involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions.

Question 26

explain why the plasmids and bacterial cells are mixed together in a medium containing calcium ions during transformation.

Answer 26

the calcium ions make the bacterial membrane more permeable, allowing the plasmids to pass through the cell-surface membrane into the cytoplasm.

Question 27

only a few bacterial cells (as little as 1%) take up the plasmids when the two are mixed together. explain how you could identify which bacterial cells have taken up the plasmid.

Answer 27

• bacteria have evolved mechanisms for resisting the effects of antibiotics by producing an enzyme that breaks down the antibiotic before it can destroy the bacterium.
• the genes for the production of these enzymes are found in the plasmids.
• the bacterial cells are grown on a medium that contains an antibiotic.
• bacterial cells that have taken up the plasmids will acquire the genes for antibiotic resistance. they will be able to break down the antibiotic and therefore survive.
• the bacterial cells that have not taken up the plasmids will not be resistant to the antibiotic and therefore die.

Question 28

what is this technique called?

Answer 28

replica plating.

Question 29

another way of identifying whether a gene as been taken up by bacterial cells is through the use of marker genes. this involves using a second separate, gene on the plasmid. other than antibiotic resistance, give two reasons why this second gene may be easily identifiable.

Answer 29

• it may produce a fluorescent protein, which can be easily seen.
• it may produce an enzyme whose action can be identified.

Question 30

give two advantages of in vivo gene cloning.

Answer 30

almost no risk of contamination - this is because a gene that has been cut by the same restriction endonuclease can match the sticky ends of the opened up plasmid. contaminant DNA will therefore not be taken up by the plasmid.

specific genes are cut out - the culturing of transformed bacteria produces many copies of a specific gene, and not just copies of the entire DNA sample.

Question 31

the polymerase chain reaction (PCR) is a method of coping fragments of DNA. give the components required for this process to occur.

Answer 31

• the DNA fragment to be copied.
• DNA polymerase.
• primers.
• nucleotides.
• thermocycler.

Question 32

DNA polymerase is an enzyme capable of joining together tens of thousands of nucleotides in a matter of minutes. state where taq polymerase is found, and explain what makes it significant from other types of DNA polymerase.

Answer 32

taq polymerase is obtained from bacteria in hot springs, so is therefore thermostable, and does not denature under high temperatures unlike most other enzymes.

Question 33

give the three stages involved in the polymerase chain reaction.

Answer 33

• separation of the DNA strand.
• annealing of the primers.
• synthesis of DNA.

Question 34

at the beginning of the polymerase chain reaction, the DNA fragments, primers, and DNA polymerase are placed in a vessel in the thermocycler, and heated to 95°C. explain why the conditions are heated to this temperature.

Answer 34

the conditions are heated to 95°C to break the hydrogen bonds between the two DNA strands, separating the DNA fragments.

Question 35

explain the significance of primers during the second stage of the PCR.

Answer 35

• the primers anneal to their complementary bases at the end of the DNA fragment.
• the primers provide the starting sequences for DNA polymerase to begin copying DNA, as DNA polymerase can only attach nucleotides to the end of an existing chain.

Question 36

during the second stage of the PCR, the reaction mixture is cooled to 55°C. this is raised to 72°C for the final stage, synthesis of DNA. explain why this rise in temperature occurs.

Answer 36

the temperature is increased to 72°C, as this is the optimum temperature for the DNA polymerase to add nucleotides along each of the separated DNA strands.

Question 37

give two advantages of in vitro gene cloning.

Answer 37

rapid - within a matter of hours, 100 billion copies of a gene can be produced using the polymerase chain reaction. in vivo cloning would take several days or weeks to produce the same quantity of DNA.

does not require living cells - all that is required for in vitro gene cloning is a base sequence of DNA that needs amplification.

Question 38

give three risks involved in using recombinant DNA technology.

Answer 38

• any manipulation of the DNA of a cell will have consequences for the metabolic pathways within that cell.
• genetically modified bacteria often have antibiotic resistant marker genes added. these bacteria may spread antibiotic resistance to harmful bacteria.
• long-term effects of the artificial selection of ‘desired’ genes could reduce the genetic variation in a population, and eventually species.

Question 39

explain the role of a DNA probe, and give the two most commonly used DNA probes.

Answer 39

• a DNA probe is a short, single-stranded length of DNA that has a label attached in order to make it easily identifiable.
• DNA probes are used to identify particular alleles of genes.
• the two most commonly used DNA probes are radioactively labelled probes, and fluorescently labelled probes.

Question 40

explain how DNA probes identify the particular alleles of genes.

Answer 40

• a DNA probe, with base sequences complimentary to that of base sequence of the DNA that makes up the allele of the desired gene, is manufactured.
• the DNA sample that is being tested is treated to separate the double strands.
• the separated strands are mixed in with the probe, which binds to the complementary base sequence on one of the DNA strands (DNA hybridisation)
• the site at which the probe binds can be identified by the radioactivity or fluorescence that the probe emits.

Question 41

what is genetic screening?

Answer 41

genetic screening is the process of testing an individual to find changes in the base sequence of their DNA to identify mutations that could cause genetic disorders.

Question 42

give an advantage of genetic screening.

Answer 42

• one advantage of genetic screening is personalised medicine.
• this allows doctors to provide advice and healthcare based on an individual’s genotype.

Question 43

what is genetic counselling used for?

Answer 43

genetic counselling is used to advise on the risk of passing on genetic disorders, and can be given to couples in certain risk groups who intend to have children.

Question 44

give one important aspect of genetic counselling.

Answer 44

one important aspect of genetic counselling is to research the family history of an inherited disease, and advise parents on the likelihood of it arising in their children.

Question 45

gene counselling is closely linked to genetic screening, and the screening results provide the genetic counsellor with a basis for informed discussion. in cases of cancer, give three factors genetic screening can help to detect.

Answer 45

• oncogene mutations, which determine the type of cancer that the patient has, and hence the most effective drug or radiotherapy treatment to use.
• gene changes, that predict which patients are more likely to benefit from certain treatments and have the best chance of survival.
• a single cancer cell among millions of normal cells, to identify patients at risk of relapse from certain forms of leukaemia.

Question 46

non-coding DNA bases are known as what?

Answer 46

• variable number tandem repeats (VNTRs)
• for every individual, the number and length of VNTRs has a unique pattern.

Question 47

what is gel electrophoresis used for?

Answer 47

to separate DNA fragments according to their size.

Question 48

explain how DNA fragments are separated by size using gel electrophoresis

Answer 48

• the DNA fragments are placed onto agar gel, and a voltage is applied across the gel.
• DNA is negatively charged, so the DNA fragments will move towards the positively charged current.
• the larger the DNA fragments, the more slowly they move, and the smaller the distance over which they travel.
• the smaller fragments will move further than the larger ones over a fixed period.
• in this way, DNA fragments of different lengths are separated.

Question 49

give the five main stages involved in the production of a genetic fingerprint.

Answer 49

• extraction.
• digestion.
• separation.
• hybridisation.
• development.

Question 50

explain why the agar gel is immersed in alkali during the separation stage of genetic fingerprinting.

Answer 50

the agar gel is immersed in alkali in order to separate the double strands of DNA into single strands.

Question 51

give three uses of DNA fingerprinting.

Answer 51

• paternity tests.
• forensic science.
• medical diagnosis.