The polymerase chain reaction (PCR) can be used to produce large quantities of a specific gene.

The main stages of PCR are as follows:

• DNA sample containing required gene is isolated from a DNA sample.
• The DNA mixture is heated to 95°C.
• The DNA mixture is then cooled to 65°C and primers are added.
• The DNA mixture is incubated at 72°C with DNA polymerase.

This process is repeated until the required quantity of DNA is produced.

Describe how a gene can be isolated from a sample of human DNA.

Explain why the DNA mixture is heated to 95°C.

Explain why primers must be added to the reaction mixture.

Explain the role of DNA polymerase in PCR.

The diagram below shows some of the steps involved in producing genetically modified bacteria.

In the process shown above, a plasmid is used as a vector.

What is the role of a vector?

Name and describe the role of the enzyme involved in steps 1 and 2 in the diagram above.

Name and describe the role of the enzyme involved in step 3 in the diagram above.

Step 4 results in some genetically modified bacteria.

Explain how scientists can determine which bacteria have been genetically modified in this way.

The entire human genome was sequenced in 2003.

Since then, new sequencing techniques have been developed.

In 2017, approximately 50 million kilobases could be sequenced per hour.

What is meant by a genome?

In 1980, DNA could be sequenced at a speed of approximately 500 bases per hour.

Calculate how many times faster DNA sequencing was in 2017 compared to 1980.

Explain how DNA sequencing can be used to determine the amino acid sequence of a protein.

Scientists can sequence the genome of a virus in order to develop a vaccine.

Explain how.

EcoRI is an enzyme that cuts DNA at specific base sequences.

The action of EcoRI is shown in the diagram below.

The arrows represent the position where the DNA strand is cut.

What name is given to enzymes that act in the same way as EcoRI?

EcoRI produces DNA fragments with sticky ends.

Use the diagram above to give the base sequence of one of these sticky ends.

Explain the purpose of sticky ends when producing recombinant DNA.

Once recombinant DNA has been produced, it can be amplified using the polymerase chain reaction (PCR).

Describe and explain how PCR can produce large quantities of recombinant DNA.

A strain of bacteria known as Bacillus thuringiensis (Bt) produce a protein that is toxic to insects.

Scientists have successfully genetically modified soybean plants to include the gene that encodes this toxic protein.

The genetically modified plants contain the gene coding for the toxic protein, a promoter, and a herbicide resistant gene that is used as a marker.

Describe how scientists could remove the gene from bacterial DNA.

The gene was injected into isolated cells of soybean plants which were then cloned.

The cloned cells then grew into new plants.

What is the advantage of inserting the gene into isolated cells rather than into cells of a whole plant?

Suggest how the herbicide resistance gene was used as a genetic marker.

A pesticide containing the toxic protein can be sprayed directly onto soybean plants.

Explain one advantage to farmers of growing genetically engineered soybean plants rather than spraying the plants with pesticide.

Suggest one reason why some people are concerned that the Bacillus thuringiensis gene might get transferred to wild plants.

A researcher used electrophoresis to identify the presence of specific genes in three individuals.

His results are shown in the diagram below.

Before DNA fragments are loaded into wells, the DNA samples from each individual must be broken down into smaller fragments.

Describe how the researcher could break down the DNA samples into small fragments.

Explain how electrophoresis separates the fragments of DNA.

Describe how DNA can be visualised after electrophoresis has been completed.

Explain how the ladder enables the sizes of the different DNA fragments to be determined.

Scientific researchers can manufacture large quantities of human insulin using genetically modified bacteria.

The first step in this process is to isolate mRNA from human pancreas cells.

This mRNA is then used to produce DNA coding for the protein insulin.

Suggest why it is more useful to start with mRNA rather than DNA.

Describe how the researchers can obtain DNA from mRNA.

After DNA has been isolated from human pancreas cells, it is used to produce genetically modified bacteria.

These bacteria can produce human insulin for use in the treatment of type 1 diabetes.

Describe how the insulin gene is inserted into bacterial cells.

Some researchers use an antibiotic resistance gene to act as a marker in the process of producing genetically modified bacteria.

Suggest how an antibiotic resistance gene enables researchers to identify genetically modified bacteria.

Some people have raised concerns about the use of genetically modified bacteria.

Give one concern that these people may have.

Researchers investigated how gene expression differs in two strains of virus.

Their investigation involved the following steps:

1 - The researchers extracted mRNA from cells infected by each strain of virus.

2 - This mRNA was then used to produce single-stranded DNA.

3 - The DNA was attached to a fluorescent dye.

4 - These lengths of single-stranded DNA were used as gene probes and fixed onto a microarray DNA chip.

5 - mRNA extracted from infected cells only bound to gene probes that were complementary, causing these probes to fluoresce.

6 - The parts of the microarray chip that give off the most fluorescence reveal which genes are the most active.

Name the enzyme that can be used to convert mRNA to single-stranded DNA.

Explain how the locations of fluorescent spots on the microarray chip identify which genes are most active.

DNA sequencing can be used to determine the genomes of these virus strains.

This information can be helpful for researchers developing a vaccine against diseases caused by these viruses.

Describe how sequencing can be used to increase the effectiveness of a vaccination programme against a virus.

Bioinformatics is also helpful when developing a vaccination programme.

Suggest how.

Cystic fibrosis is a genetic disease that has the potential to be treated using gene therapy.

People with cystic fibrosis have two copies of a recessive allele, causing their cells to produce insufficient quantities of the CFTR protein.

The allele that codes for the functioning protein can be inserted into the DNA of cystic fibrosis patients.

However, current research shows that the effect of gene therapy might only last for a few months.

Suggest how the insertion of a new gene into a chromosome may affect the functioning of other genes in the same chromosome.

Current research into the treatment of cystic fibrosis also involves the use of induced pluripotent stem cells (iPSCs).

Suggest three advantages of using iPSCs rather than gene therapy to treat cystic fibrosis.

Huntington’s disease is a dominant disorder affecting nerve cells in the brain.

It is caused by a dominant allele that codes for the protein huntingtin.

Scientists do not yet know how the production of this protein causes the symptoms of Huntington’s disease.

Suggest why gene therapy will not work as a treatment for Huntington’s disease.

DNA profiling can be used to analyse the genetic relationship between individuals.

Before DNA profiling can begin, the DNA needs to be purified.

Explain why a protease enzyme is added to the mixture during DNA purification.

Explain why DNA profiling only uses selected sections of non-coding human DNA.

A paternity test has been carried out to find out the genetic father of a child.

The results of the genetic profiles for the child and the two suspected fathers are shown in the diagram below.

Which male, X or Y, is most likely to be the genetic father of the child?

Explain your answer.