3.8.4.1 - RECOMBINANT DNA TECHNOLOGY

Question 1

short tandem repeats (STRs) are short sequences of DNA, usually 2 to 7 base pairs. STR are repeated a number of times, one after another.

for example, the STR D5S818 is made up of AGAT repeated 7 to 16 times.

the repeated sequences in STRs are common to all humans. due to variation in the number of repeats, STRs can be used in genetic fingerprinting.

describe how STRs could be removed from a sample of DNA (2)

Answer 1

1. use restriction endonucleases
2. cut DNA at specific base sequences

Question 2

short tandem repeats (STRs) are short sequences of DNA, usually 2 to 7 base pairs. STR are repeated a number of times, one after another.

for example, the STR D5S818 is made up of AGAT repeated 7 to 16 times.

the repeated sequences in STRs are common to all humans. due to variation in the number of repeats, STRs can be used in genetic fingerprinting.

genetic fingerprinting using STRs requires amplification of the STRs using the polymerase chain reaction (PCR). the short base sequences either side of a specific STR are known.

explain the importance of knowing these base sequences in PCR (2)

Answer 2

1. for primers
2. to produce a complementary base sequence OR primers provide starting sequence for DNA polymerase OR primers stop original DNA strands re-joining

Question 3

name the type of enzyme used to create cDNA (1)

Answer 3

reverse transcriptase

Question 4

plasmids are often used as vectors in genetic engineering

what is the role of a vector (1)

Answer 4

transfer/carry genes from one organism to another

Question 5

describe the role of restriction endonucleases in the formation of plasmids that contain donor DNA (2)

Answer 5

1. cut cut open plasmid
2. cut donor DNA to remove gene
3. cut donor DNA and plasmid using the same enzyme
4. sticky ends

Question 6

describe the role of DNA ligase in the production of plasmids contains donor DNA (1)

Answer 6

annealing/ phosphodiester bonds

Question 7

the polymerase chain reaction (PCR) can be used to produce large quantities of DNA.

describe how the PCR is carried out (6)

Answer 7

1. DNA heated to 95C
2. strands separate (hydrogen bonds break)
3. cooled to 55C
4. allows primers to bind (hydrogen bonds reform) (annealing)
5. nucleotides attach
6. by complementary base pairing
7. temperature 72C
8. taq polymerase joins nucleotides together
9. cycle repeats

Question 8

Many copies of the isolated gene were required. Name the process used in a laboratory to produce many copies of DNA from a small amount. (1)

Answer 8

polymerase chain reaction

Question 9

explain the reason for each of the following in the polymerase chain reaction (3)

1. DNA is heated to 95C
2. DNA taq polymerase used is heat stable
3. the mixture is cooled to 55C

Answer 9

1. to separate polynucleotide strands
2. not denatured at 95C
3. for binding of primers

Question 10

suggest one use of the polymerase chain reaction (1)

Answer 10

replication of DNA from a crime scene

Question 11

give two ways in which the PCR differs from the process of transcription (2)

Answer 11

1. transcription uses RNA polymerase
2. RNA nucleotides/uracil
3. in transcription one template strand, in PCR both

Question 12

whole potato plants can be produced from genetically identical potato cells grown in a tissue culture.

use your knowledge of genes to suggest how different cells, such as leaf and root cells, can develop from genetically identical cells (2)

Answer 12

1. different genes are expressed
2. producing different enzymes/proteins

Question 13

use your knowledge of enzymes to explain why restriction enzymes only cut at specific restriction sites (2)

Answer 13

1. cut at a specific sequence
2. results in a different shape of active site
3. therefore only that sequence will fit the active site of the enzyme

Question 14

explain how modifies plasmids are made by genetic engineering and how the use of markers enable bacteria contains these plasmids to be detected (6)

Answer 14

1. isolate wanted gene
2. using restriction endonuclease
3. to form sticky ends in DNA and plasmid
4. use ligase to join gene to plasmid
5. also include marker gene - antibiotic resistance
6. culture with bacteria to allow uptake of plasmids
7. use of heat shock to enhance uptake
8. then plate onto medium where the marker gene will be expressed
9. remaining bacteria have wanted gene

Question 15

what is gene therapy? (1)

Answer 15

introduction of healthy gene / replacement of defective gene

Question 16

give two advantages of using viruses in gene therapy (2)

Answer 16

1. they can enter cells
2. targets specific cells
3. replicates in cells

Question 17

scientists manufactured large quantities of human insulin using genetic engineering. they started by isolating mRNA from pancreas cells.

from this they produced DNA which coded for insulin

suggest two reasons why it was better to start with mRNA from the pancreas cells rather than with the DNA from these cells (2)

Answer 17

1. amount of mRNA > amount of DNA
2. introns removed from mRNA

Question 18

explain the function of primers (2)

Answer 18

allow hydrogen bond reformation

Question 19

explain how sticky ends are useful in genetic engineering (2)

Answer 19

1. joining two pieces of DNA
2. by complementary base pairing

Question 20

explain the role of reverse transcriptase in reverse transcriptase polymerase chain reaction (1)

Answer 20

produces cDNA using mRNA

Question 21

explain the role of DNA polymerase in PCR (1)

Answer 21

joins nucleotides to produce strand of DNA

Question 22

explain why when amplifying RNA fragments, any DNA in the sample is hydrolysed by enzymes before the sample is added to the reaction mixture (2)

Answer 22

1. to remove any DNA present
2. as this DNA would be amplified/replicated

Question 23

suggest why DNA replication stops in the PCR (1)

Answer 23

limited number of nucleotides

Question 24

scientists have used the PCR method to detect the presence of different RNA viruses in patients suffering from respiratory diseases.

explain why the scientists produced a variety of primers for this procedure (2)

Answer 24

1. base sequences differ (because they are different viruses)
2. so different complementary primers required

Question 25

the geneticist concluded that it would be faster to create the HGH gene using a gene machine rather than by using reverse transcriptase to convert mRNA for HGH into cDNA suggest why the geneticist reached this conclusion (1)

Answer 25

faster to use gene machine than all the enzyme catalysed reactions involving reverse transcriptase

Question 26

suggest why the plasmids were injected into the eggs of silkworms rather than into silkworms (2)

Answer 26

1. so gene gets into all of the cells of the silkworm (as it will divide by mitosis) 2. so it will get into the cells that make the silk

Question 27

what would the scientists have inserted into the plasmid along with the spider gene to ensure that the spider gene was only expressed in the silk glands of the silkworms (1)

Answer 27

promoter region/gene

Question 28

suggest two reasons why it was important that the spider gene was expressed only in the silk glands of the silkworms (2)

Answer 28

1. so that the protein can be harvested 2. fibres in other cells may cause harm

Question 29

the desired gene in this diagram was from an insect. in stage 6, the plant containing this gene was able to use it to synthesise an insect protein. the plant is able to synthesise insect protein. explain why this is possible (3)

Answer 29

1. genetic code is universal/triplets in DNA always code for the same amino acid 2. insect DNA can be transcribed 3. can be translated as this process/mechanism is the same in all organisms

Question 30

what is meant by a non-coding base sequence? (1)

Answer 30

DNA that does not code for amino acid/RNA

Question 31

the scientists wanted to know on which chromosome the alles R and r were 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 (2)

Answer 31

1. chromosomes visible 2. can see which chromosome the DNA probe attached to

Question 32

many attempts to produce transgenic animals have failed. very few live births result from the many embryos that are implanted. suggest one reason why (2)

Answer 32

1. mutation may occur **OR** DNA may be damaged 2. may interfere with gene expression 3. embryo/antigens foreign 4. embryo rejected/attacked by immune system

Question 33

it is important that scientists still report the results from failed attempts to produce transgenic animals. explain why (2)

Answer 33

1. saves time/money for others 2. so the same work is not repeated/methods can be compared/improved/amended

Question 34

scientists made an artificial gene which codes for insulin. they put the gene into a virus which was then injected into rats with type 1 diabetes. explain why this form of gene therapy may be less effective in treating rats that have type 2 diabetes (1)

Answer 34

type 2 diabetes still produce insulin

Question 35

scientists made an artificial gene which codes for insulin. they put the gene into a virus which was then injected into rats with type I diabetes. the virus was harmless to the rats but carried the gene into the cells of the rats. the treated rats produced insulin for up to 8 months and showed no side-effects. the scientists measured the blood glucose concentrations of the rats at regular intervals. while the rats were producing the insulin, their blood glucose concentrations were normal. research workers have suggested that treating diabetes in humans by this method of gene therapy would be better than injecting insulin. evaluate this suggestion (4)

Answer 35

1. avoids (pain of) injections 2. longer lasting/permanent 3. less need to measure blood sugar 4. less restriction on diet 5. rats are different to humans 6. may have side effects on humans 7. the insulin may be rejected by the body

Question 36

cut plasmids and lengths of foreign DNA can join. what features of their ends allow them to join? (2)

Answer 36

sticky ends

Question 37

why are primers required in PCR (1)

Answer 37

to allow DNA polymerase to attach/mark start and end of of the sequence to be copied

Question 38

suggest why two different primers are required (1)

Answer 38

because the sequences at the ends of the target sequence are different

Question 39

suggest how cells with the resistance gene may be selected. (2)

Answer 39

1. expose cells to the fungus/antibiotic etc 2. non-resistant ones die, resistant ones survive **OR** 1. identify by adding marker gene/gene probe 2. marker probe - description of positive result e.g. radioactivity or fluorescence

Question 40

why are primers added during the PCR? (1)

Answer 40

1. to mark beginning/ends of the parts of the DNA needed 2. for attachment of enzymes or nucleotides 3. keeps strands apart

Question 41

what is the advantage of the enzyme used in the PCR being thermostable? (2)

Answer 41

1. would not be denatured 2. must withstand high temps of 95C

Question 42

complete the following definitions (2) the genome is… the proteome is…

Answer 42

1. genome is the complete set of genes in a cell/organism 2. proteome is the range of proteins a cell can produce **OR** the genome can code for

Question 43

recombinant DNA technology can involve the transfer of fragments of human DNA into bacteria. the bacteria are then used to produce human proteins. give two reasons why bacteria are able to use human DNA to produce human proteins (2)

Answer 43

1. the genetic code is universal **OR** the same triplets/cordons code for the same amino acids in all species 2. the mechanism of transcription is universal 3. the mechanism of translation is universal

Question 44

suggest and explain one reason why bacteria might not be able to produce every human protein (1)

Answer 44

1. cannot splice pre-mRNA so cannot remove introns 2. do not have Golgi so cannot modify proteins

Question 45

describe and explain how the PCR is used to amplify a DNA fragment (4)

Answer 45

1. requires DNA fragment, DNA polymerase, DNA nucleotides and primers 2. heat to 95C to break hydrogen bonds 2. reduce temperature to 55C to allow primers to bind to DNA strands 4. increase temperature, DNA polymerase joins nucleotides and repeat