Biology Unit 5 Chapter 16 - DNA Technology

Explain the reason for adding each of the following to the tubes. (i) DNA polymerase (ii) Primers (2)

(i) Joins nucleotide together; (ii) Enables sequencing to start

When the scientists digested another of the recombinant plasmids with BamH1, they obtained three fragments. How many BamH1 restriction sites are there in the unknown piece of DNA? (1)

2

How many DNA molecules will have been produced from one molecule of DNA after 6 complete cycles? (1)

64

Describe the role of DNA ligase in the production of plasmids containing donor DNA. (1)

Backbones joined

Two bands are usually seen for each person tested. Suggest why only one band was seen for Person L. (1)

CAG fragments are the same length

Suggest why panda DNA is found in faeces. (1)

Cells from panda are in faeces

Explain why the labelled DNA probe could be used to find out whether the haplotypes were the same. (2)

Complimentary base sequence; You would see if it binds to both haplotypes

Why are these methods not always effective? (3)

Even when gene is successfully delivered to epithelial cells, very few are expressed; The gene and the vector may cause an immune response so it is often rejected; Effect is short-lived because somatic cells are not passed onto offspring and die so need to be replaced

Use Figure 2 to determine the sequence of nucleotides in this sample of DNA. (1)

GAAGTCTCAG

In which two ways may gene therapy be used to treat cystic fibrosis? (2)

Gene replacement - replacing defective gene with healthy gene; Gene supplementation - healthy gene added alongside defective gene, which is dominant so masks effects of the recessive allele

What is the role of DNA polymerase? (1)

Joins DNA nucleotides

One of the fragments that the scientists obtained was described as "very large". What is represented by this very large fragment? (1)

Most of plasmid and the rest of the unknown DNA

When plasmids are reintroduced into bacterial cells, not all the bacterial cells will possess the desired DNA fragments. Suggest the two main reasons for this. (2)

Only a few bacterial cells take up the plasmids when they are mixed together; Some plasmids will have closed up without incorporating the DNA fragment

What makes the DNA fragments visible on the autoradiograph? (1)

Radioactive primer

What are the two types of DNA probes? (2)

Radioactively labelled probes; Fluorescently labelled probes

(Refer to exam q) Gene therapy is used to treat the genetic disorder,ADA deficiency. Affected individuals are unable to produce the enzyme adenosine deaminase (ADA). Without this enzyme, T lymphocytes, a type of white blood cell, cannot provide immunity to infection. The diagram shows the processes involved in the treatment of ADA deficiency by gene therapy. What is meant by gene therapy? (1)

Replacement of defective gene

Suggest one use of the polymerase chain reaction. (1)

Replication of DNA from crime scene

Describe how a gene can be inserted into a vector by using restriction endonucleases. (5)

Restriction endonucleases cut DNA at recognition site; A plasmid is cut at its gene for antibiotic resistance using the same restriction endonuclease; The sticky ends of the DNA fragments are complementary to the sticky ends of the cut plasmid; DNA fragments and open vectors are mixed with DNA ligase; Ligase joins the phosphate-sugar frameworks together

mRNA can be converted to cDNA. Name the enzyme used in this process. (1)

Reverse transcriptase

How are antibiotic-resistance markers used to find out which bacterial cells have taken up the desired gene? (6)

The bacterial cells that survived the treatment with the first antibiotic took up the plasmid; These cells are grown on an agar plate in colonies; A sample of each colony is tranferred onto a replica agar plate in exactly the same position on the plate as the original colonies; The replica plate contains the second antibiotic; The colonies killed by the antibiotic are the ones that have taken up the required gene; These colonies are located on the original plate

About twenty percent of the DNA produced by the PCR is copied inaccurately. Suggest and explain why it is not safe to use the PCR to clone the CFTR gene for use in treating cystic fibrosis. (4)

The percentage risk is too high for human application; If the sequence of amino acids changes; A non-functional protein may be produced; Which will be harmful

What is the role of a vector in in vivo cloning? (1)

Transfers DNA from one organism into another

(Refer to June 2010 paper) There are wolves in many European countries. Scientists investigated the genetic diversity of these wolves. They collected samples of DNA from the mitochondria of wolves from different countries. For each sample they identified which haplotypes were present in the DNA. A haplotype is a particular sequence of bases on DNA. Mutations can produce new haplotypes. The scientists wanted to find out whether one of the haplotypes in the Portuguese wolves was the same as one of those in the Spanish wolves. They used a restriction endonuclease, electrophoresis and a labelled DNA probe. For what purpose did they use (i) the restriction endonuclease (ii) electrophoresis?

(i) To cut the DNA (ii) To separate the pieces of DNA

How is complementary DNA made using reverse transcriptase? (4)

A cell that readily produces the required protein is selected; The relevant mRNA is extracted; mRNA acts as a template on which a complementary single-stranded copy of DNA is formed using reverse transcriptase; DNA polymerase joins complementary free nucleotides onto the single DNA strand; Forms a double-stranded DNA molecule with the required gene

What is a DNA probe? (2)

A short, single-stranded section of DNA; that has a label attached to make it easily identifiable

A sample of DNA analysed by this technique had the following nucleotide base sequence. T G G T C A C G A Give the base sequence of the shortest DNA fragment which would be produced in Tube 2. (1)

AC

When genes are introduced into cells, antibiotic resistance genes are often added as well. Explain the reason for this. (2)

Acts as a marker gene; Allows identification of cells with the new gene

The jellyfish gene attached to the human Factor IX gene (Stage 2) codes for a protein that glows green under fluorescent light. Explain the purpose of attaching this gene. (2)

Acts as a marker gene; Shows that the human gene has been taken up

How is the normal CFTR gene introduced into a cystic fibrosis sufferer through somatic-cell gene therapy using a harmless virus? (5)

Adenovirus made harmless by interfering with a gene involved in their replication; Adenovirus grown in epithelial cells, along with plasmids that contain the normal CFTR gene; CFTR gene becomes incorporated into the DNA of the adenovirus; Adenovirus isolated from epithelial cells and purified; Adenovirus introduced into nostrils of patient and inject their DNA into the epithelial cells of the lungs

Suggest one benefit and one disadvantage of using genetically modified herbicide-resistant crop plants together with the relevant herbicide. Explain your answer. (2)

Advantage: Crop yield is greater because the herbicide kills the weeds that are competing for light, water and minerals but not the crop; Disadvantage: The herbicide might accumulate further up in the food chain and might be toxic to other organisms

What information is given in genetic counselling? (3)

Advice on how likely it is that a certain disease will arise in parents' offspring; The emotional, psychological, economic and social consequences of a disease; The best course of treatment

Explain how genetic fingerprinting allows scientists to identify the father of a particular panda cub. (2)

All bands in cub which dont come from mother; Must be in fathers DNA fingerprint

How can you find out which bacterial cells have taken up the plasmids? (4)

Bacterial cells are grown on a medium that contains an antibiotic; Bacterial cells that have taken up the plasmids will have acquired the gene for resistance of the antibiotic; These bacterial cells are able to break down the antibiotic and survive; Cells that have not taken up the plamid will not be resistant to antibiotic and die

Why are the bacterial cells that are killed by the second antibiotic the cells that have taken up the desired gene? (2)

Because the antibiotic resistance gene was cut to incorporate the desired gene; So no longer produces the enzyme that breaks down the second antibiotic

How is the normal CFTR gene introduced into a cystic fibrosis sufferer through somatic-cell gene therapy using lipid molecules? (5)

CFTR genes isolated from tissue and inserted into plasmids; Plasmids reintroduced into bacterial cells and gene markers are used to detect which bacteria have taken up plasmids with gene; Bacteria cloned; Plasmids extracted from bacteria and wrapped in lipid molecules to form liposomes; Liposomes sprayed into nostrils and pass across phospholipid bilayer of epithelial cell-surface membranes

Explain how the presence of an altered protein in people with cystic fibrosis results in the production of very thick and sticky mucus, and how this accounts for the symptoms of this disorder. (5)

CFTR; blocks outward passage of chloride ions; so water is retained in the cell; Therefore mucus is unable to be removed in the lungs so infection is more likely; Mucus causes narrowing of air passages so breathing is more difficult

The ADA gene is inserted into a virus. Give two advantages of using a virus in gene therapy. (2)

Can inject DNA into cells; Targets specific cells

(Refer to June 2013 paper) A husband and wife wanted to know whether they were carriers of the mutated form of a gene. This mutation is a deletion that causes a serious inherited genetic disorder in people who are homozygous. A geneticist took samples of DNA from the husband and the wife. He used a DNA probe to look for the deletion mutation. The DNA probe was specific to a particular base sequence in an exon in the gene. Exons are the coding sequences in a gene. The geneticist compared the couple's DNA with that of a person known not to carry this mutation. The chart shows the geneticist's results. The geneticist told the couple they were both carriers of the mutated gene. Explain how he reached this conclusion. (3)

Carriers are heterozygous; Both have DNA that binds to about half of the amount of probe that non-carrier does; Probe binds to healthy allele

The scientists' hypothesis at the start of the investigation was that crop plants genetically engineered to produce GB would become more resistant to high environmental temperatures. The scientists developed this hypothesis on the basis of previous research on crops that are grown in hot climates. Suggest how the scientists arrived at their hypothesis. (2)

Crop plants cited in this research make GB; So assumed making plants produce GB makes them resistant to high temperatures

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

Cut open plasmid; Cut donor DNA, to remove gene

Research scientists can increase the nutritional value of potatoes by genetically engineering potato plants. A gene which results in increased protein production has been removed from cells of an amaranth plant and inserted into cells of a potato plant. Describe how a gene could be removed from cells of an amaranth plant and inserted into cells of a potato plant. (6)

Cut out gene using an endonuclease; The endonuclease cuts a DNA double strand at a specific sequence of bases called a recognition site; Use the same enzyme to cut; a plasmid; The complementary bases are fixed by the enzyme DNA ligase; The vector is then introduced to the plant cell by a harmless virus injecting the DNA

Explain why the primers used in the PCR will bind to panda DNA, but not to DNA from bacteria or bamboo. (2)

DNA has a specific base-sequence; Primers bind to DNA by complementary base-pairing

The polymerase chain reaction (PCR) can be used to produce large quantities of DNA. Describe how the PCR is carried out. (6)

DNA heated to 90 - 95°C; causing the strands to separate; It is then cooled to 55°C which causes primers to bind; Temperature increased to 72°C and the nucleotides attach to the strand; by complementary base pairing; DNA polymerase joins the nucleotides together

(Refer to exam q) Use information in the passage and your own biological knowledge to answer the questions. Describe how genetic fingerprinting may be carried out on a sample of panda DNA. (7)

DNA is cut using restriction endonucleases; Gel electrophoresis is used; To separate DNA fragments according to length; These are transferred to a nylon membrane; DNA probe is applied; To radioactively label the DNA; The membrane is placed onto an X-ray film which is exposed

How are DNA probes used to locate specific genes? (4)

DNA probe has bases that are complementary to the portion of DNA sequence that makes up the required gene; DNA strands are separated; Separated DNA strands and probe are mixed in DNA hybridisation, and probes join to complementary DNA bases; The site at which the probe binds is identified by its radioactivity/fluorescence

What is cystic fibrosis? (5)

Deletion mutation causes an amino acid to be left out of the CFTR protein; Tertiary structure of protein changes; Protein unable to trasport chloride ions across epithelial membrane; So water does not leave cell into mucus layer by osmosis; Resulting in sticky mucus

(Refer to exam q) A different sample of DNA was then analysed. The DNA fragments from the four tubes were separated in a gel by electrophoresis and analysed by autoradiography. Figure 2 shows the banding pattern produced. Explain why the DNA fragments move different distances in the gel. (1)

Different lengths

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

Embryo is foreign; Embryo is attacked by immune system

What is the role of a primer in this process? (1)

Enables sequencing to start

(Refer to June 2012 paper) Haemophilia is a genetic condition in which blood fails to clot. Factor IX is a protein used to treat haemophilia. Sheep can be genetically engineered to produce Factor IX in the milk produced by their mammary glands. The diagram shows the stages involved in this process. Name the type of enzyme that is used to cut the gene for Factor IX from human DNA (Stage 1) . (1)

Endonuclease

Suggest two advantages of in vitro gene cloning. (2)

Extremely rapid, useful when there is a minute amount of DNA available, e.g. at a crime scene, so no loss of time; Does not require living cells, only a DNA base sequence

The promoter DNA from sheep (Stage 3) causes transcription of genes coding for proteins found in sheep milk. Suggest the advantage of using this promoter DNA. (2)

Factor IX present in milk; Do not need to kill sheep to obtain Factor IX

Sheep have been genetically engineered to produce alpha-1-antitrypsin which is used to treat cystic fibrosis. Use your knowledge of this process to explain one argument for and one against using sheep in this way. (2)

For: The gene is only active in mammary cells; Against: Long term effects not known

(Refer to June 2013 paper) The diagram shows the base sequence on DNA where a restriction endonuclease cuts DNA. Use evidence from the diagram to explain what is meant by a palindromic recognition sequence on DNA. (1)

GGATCC same as CCTAGG in opposite direction

Individuals who have been treated by this method of gene therapy do not pass on the ADA gene to their children. Explain why. (1)

Gamete cells do not contain ADA gene

What is the difference between germ-line and somatic-cell gene therapy? (4)

Germ-line: Involves replacing/supplementing the defective gene in the fertilised egg; Ensures that all cells, as well as cells of offspring, will develop normally; Somatic-cell: Targets just affected tissues; Treatment needs to be repeated periodically as cells are constantly dying

Explain how two parents, neither of whom suffers from cystic fibrosis, might have a child with the disease. (3)

If both parents are heterozygous for the CFTR gene, then each would carry one dominant and one recessive allele; They would not suffer as they have the dominant allele; If an offspring inherits one recessive allele from each parent, they would suffer from CF

What are the two ways in which DNA fragments can be cloned? (2)

In vivo, transferring the fragments to a host cell using a vector; In vitro, using the polymerase chain reaction

State three ways in which genetic modification of organisms has benefited humans? (3)

Increased the yield from animals and crop plants; Improved nutrient content of foods; Produced vaccines

How have tomatoes been genetically modified to prevent softening? (4)

Insertion of a gene; which has a complementary base sequence to the gene which produces the softening enzyme; The two mRNA strands join, preventing the mRNA from the original gene being translated; Softening enzyme therefore not produced

The DNA probe the geneticist used was for an exon in the DNA, not an intron. Explain why. (3)

Introns are not translated; Introns do not code for amino acids; Mutations of only exons affect amino acid sequences

Suggest four risks of recombinant DNA technology. (4)

Is the cost of genetic engineering justified, or would the money be better used to fight hunger and poverty?; The ability to manipulate genes may get into the wrong hands, so individuals, groups or goverments may use this to achieve power; Unsure of long-term consequences; Reduced genetic variety and biodiversity

(Refer to June 2011 paper) Scientists used restriction mapping to investigate some aspects of the base sequence of an unknown piece of DNA. This piece of DNA was 3 000 base pairs (bp) long. The scientists took plasmids that had one restriction site for the enzyme Kpn1 and one restriction site for the enzyme BamH1. They inserted copies of the unknown piece of DNA into the plasmids. This produced recombinant plasmids. The diagram shows a recombinant plasmid. When the scientists digested one of the recombinant plasmids with Kpn1, they obtained two fragments. One fragment was measured as 1 000 bp. The other fragment was described as "very large". What does this show about the base sequence of the unknown piece of DNA? (2)

It has the restriction site cut by Kpn1; Once

(Refer to exam q) There are many different restriction endonucleases. Each type cuts the DNA of a plasmid at a specific base sequence called a restriction site. The diagram shows the position of four restriction sites, J, K, L and M, for four different enzymes on a single plasmid. The distances between these sites is measured in kilobases of DNA. The plasmid was cut using only two restriction endonucleases. The resulting fragments were separated by gel electrophoresis. The positions of the fragments are shown in the chart below. Which of the restriction sites were cut? Explain your answer. (2)

L and M; The length of the fragments were 64 and 36 kilobases

Scientists need to take precautions when they carry out restriction mapping. They need to make sure that the enzyme they have used has completely digested the DNA. One check they may carry out is to add the sizes of the fragments together. How could scientists use this information to show that the DNA has not been completely digested? Explain your answer. (2)

Large pieces of DNA present; Add up to more than the total length of original DNA

Some scientists are concerned about the use of antibiotic resistance genes in genetic engineering. Suggest why. (2)

May pass to bacteria; Pathogens may become resistant and unable to treat disease

Suggest four advantages of recombinant DNA technology. (4)

Microorganisms can be modified to produce a range of substances, e.g. antibiotics, hormones and enzymes, that are used to treat disease; Genetically modified crops can be modified to make them more tolerant to environmental extremes, so they can be grown in various places; Gene therapy can be used to cure certain genetic disorders, such as cystic fibrosis; Genetically modified animals can be modified to increase the yield of milk, meat etc.

When a modified nucleotide is used to form a complementary DNA strand, the sequencing reaction is terminated. Suggest how this sequencing reaction is terminated. (1)

Modified nucleotide does not fit DNA polymerase

What is the cause of cystic fibrosis? (2)

Mutant recessive allele; Deletion mutation in which A-A-A are missing

(Refer to June 2013 paper) Some species of crop plant produce a substance called glycinebetaine (GB). Scientists transferred the gene for GB into a species of crop plant that does not normally produce GB. These genetically modified plants then produced GB. The scientists grew large numbers of the same crop plant with and without the gene at different temperatures. After 3 days, they found the increase in dry mass of the plants. Figure 1 shows their results. Describe the effect on growth of transferring the gene for GB into this plant. (2)

No effect at 25 degrees; Keeps growing up to 35 degrees, more than without GB

(Refer to June 2012 paper) Huntington's disease is a genetic condition that leads to a loss in brain function. The gene involved contains a section of DNA with many repeats of the base sequence CAG. The number of these repeats determines whether or not an allele of this gene will cause Huntington's disease. Scientists took DNA samples from three people, J, K and L. They used the polymerase chain reaction (PCR) to produce many copies of the piece of DNA containing the CAG repeats obtained from each person. They separated the DNA fragments by gel electrophoresis. A radioactively labelled probe was then used to detect the fragments. The diagram shows the appearance of part of the gel after an X-ray was taken. The bands show the DNA fragments that contain the CAG repeats. Only one of these people tested positive for Huntington's disease. Which person was this? Explain your answer. (2)

Person K; As has band that travelled the shortest distance so has largest fragment and therefore highest number of CAG repeats

(Refer to exam q) One technique used to determine the sequence of nucleotides in a sample of DNA is the Sanger procedure. This requires four sequencing reactions to be carried out at the same time. The sequencing reactions occur in four separate tubes. Each tube contains • a large quantity of the sample DNA • a large quantity of the four nucleotides containing thymine, cytosine, guanine and adenine • DNA polymerase • radioactive primers A modified nucleotide is also added to each tube, as shown in Figure 1. A large quantity of the DNA sample is required for this procedure. Name the reaction used to amplify small amounts of DNA into quantities large enough for this procedure. (1)

Polymerase chain reaction

(Refer to BYB2 June 2005 paper) Cystic fibrosis can be caused by any one of several mutant alleles of the cystic fibrosis gene. The most common of these mutant alleles accounts for about 70% of cases of cystic fibrosis. The use of gene probes can identify individuals carrying this allele. Gene probes are single strands of DNA which are radioactively labelled. They have a base sequence that is complementary to a mutant allele. The main stages in using a gene probe are shown in the diagram. Using the information given, explain how the use of a gene probe could enable the presence of a mutant allele of the cystic fibrosis gene to be detected. (4)

Probe will attach to the mutant allele; It only attaches to one DNA strand; as a result of complementary base pairing; This is radioactivity detected on film if the mutant allele is present

To make the DNA probe, the geneticist had to find the base sequence of the normal gene. Once he had copies of the gene, what methods would he use to find the base sequence of the gene? (2)

Restriction mapping; Sanger method

Give one advantage of using fluorescent gene markers rather than antibiotic gene markers. Explain your answer. (3)

Results can be obtained more easily and quickly; With antibiotic gene markers, replica plating is necessary because the cells with the required gene are killed; With fluorescent markers, the cells are not killed so replica plating is unnecessary

The diagram only shows part of the gel. Suggest how the scientists found the number of CAG repeats in the bands shown on the gel. (1)

Run fragments of known length at the same time

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

Saves time and money for others; Prevents the same errors from being made

When pandas are bred in zoos, it is important to ensure only unrelated pandas breed. Suggest how genetic fingerprints might be used to do this. (1)

Select pairs with dissimilar DNA fingerprints

How can the exact order of nucleotides on a strand of DNA be determined? (6)

Set up four test tubes, each containing single-stranded fragments of DNA to be sequenced, a labelled primer, nucleotides, one of four terminator nucleotides and DNA polymerase; The DNA fragments in each test tube will vary in length but will all end with a nucleotide that has the same base; The fragment lengths are separated using gel electrophoresis; The fragments are placed onto agar gel and a voltage is applied; The larger the fragments the more slowly they move; Photographic film is placed over the gel and is exposed by the radioactivity of each DNA fragment

The scientists measured the rate of photosynthesis in plants that produce GB and plants that do not produce GB at 25 ºC, 35 ºC and 45 ºC. The scientists concluded that the production of GB protects photosynthesis from damage by high temperatures. Use these data to support this conclusion. (1)

Significantly different - standard errors do not overlap

Scientists can separate fragments of DNA using electrophoresis. Suggest how they can use electrophoresis to estimate the number of base pairs in the separated fragments. (2)

Smaller fragments move further; Compare with distance moved by fragments of known size

Use the data from Figure 2 for plants that do not produce GB to explain the effect of temperature on changes in dry mass of the plants shown in Figure 1. (4)

Some enzymes denatured; Less photosynthesis, so fewer sugars formed; Less respiration; Less ATP for growth

State two roles of a primer used in the Sanger method of sequencing of DNA. (2)

Starts the process of DNA synthesis by making the DNA double-stranded; Carries the radioactive label for later identification of the DNA fragment produced

Explain why treatment of ADA deficiency by gene therapy must be repeated at regular intervals, whereas a single bone marrow transplant can provide a permanent cure. (2)

T lymphocytes have a limited life span; bone marrow provides continual supply of T lymphocytes

DNA from wild pandas could also be obtained from blood samples. Suggest two advantages of using faeces, rather than blood samples, to obtain DNA from pandas. (2)

Taking samples from animals causes stress to animal; Pandas are a threat to humans

Describe the process of genetic screening. (7)

The order of nucleotides on the mutated gene is determined by DNA sequencing; A fragment of DNA with complementary bases to the mutated gene is produced; A DNA probe is formed by radioactively labelling the DNA fragment; PCR used to produce more copies of the DNA probe; Probe added to single-stranded DNA fragments from individual; If the donor has mutated gene, some of their DNA fragments will have a complementary base sequence to the probe and they will join; DNA fragments will be labelled and can be distinguished by exposing X-ray film

How are plasmids reintroduced into bacterial cells? (2)

They are mixed together in a medium containing calcium ions; Calcium ions and temperature changes make the bacteria permeable to the plasmids

Explain why the PCR is carried out on the DNA from the faeces. (1)

To increase amount of DNA

T lymphocytes are produced in bone marrow. A bone marrow transplant from a genetically matched donor can provide a permanent cure for ADA deficiency. Suggest why bone marrow for a transplant is obtained from a genetically matched donor. (1)

To prevent immune response

(Refer to exam q) The polymerase chain reaction is a process which can be carried out in a laboratory to replicate DNA. The diagram shows the main stages involved in the polymerase chain reaction. Explain why DNA is heated to 95 °C. (1)

To separate the two strands

Why are gene markers necessary during in vivo cloning? (1)

To show which cells have taken up the desired gene

Give two ways in which the polymerase chain reaction differs from the process of transcription. (2)

Transcription uses RNA polymerase; and one template strand whereas PCR uses both strands

Plasmids are often used as vectors in genetic engineering. What is the role of a vector? (1)

Transfer genes from one organism to another

A strain of rice called 'golden rice' has been genetically modified to carry an extra gene. Golden rice produces more vitamin A than ordinary rice. Describe how this extra gene could be introduced into a cell of a rice plant. (1)

Use of vector

Suggest two advantages of in vivo gene cloning. (2)

Useful in introducing a gene into another organism through its use of vectors; No risk of contamination due to the specific complementary sticky ends created by restriction endonucleases