Recombinant DNA technology (A-level only)

Question 1

Recombinant DNA

Answer 1

Fragments of foreign DNA are inserted into other sections of DNA.

Question 2

Universal code

Answer 2

DNA is made from a sequence of four bases (A, T, C, G).

Every organism uses the four bases as the genetic code to produce proteins.

This means that DNA can be considered a universal code.

Question 3

Recombinant DNA cont.

Answer 3

The fact that the genetic code is universal means that any section of DNA can be taken from one organism and placed inside another.

Once the DNA has been inserted, it is then transcribed and translated to produce proteins.

Transcription and translation are also universal processes.

The process of transferring sections of DNA produces recombinant DNA.

Question 4

Fragments

Answer 4

The sections of DNA that are transferred are called fragments.

The organism that has received fragments of DNA is said to be transgenic.

Question 5

Steps in producing recombinant DNA is producing the fragments:

Answer 5

Target gene
Producing the fragment
Inserting the fragment

Question 6

Target gene

Answer 6

Recombinant DNA often involves inserting a specific gene of interest into DNA.

This gene normally encodes a protein that has useful properties.

The gene that is transferred is called the target gene.

Question 7

Producing the fragment

Answer 7

In order to transfer the target gene, it needs to be removed from the DNA in a section called a DNA fragment.

DNA fragments can be produced in three ways:
Reverse transcriptase.
Restriction endonuclease.
Using a gene machine.

Question 8

Inserting the fragment

Answer 8

Once the fragment has been produced, it can be inserted into the genome to produce a transgenic organism.

Question 9

mRNA

Answer 9

mRNA is a single-stranded molecule that is produced when a specific length of DNA (the target gene) is transcribed.

mRNA is complementary to the base sequence in the target gene.

The mRNA sequence can be used as a template for producing fragments of DNA.

Question 10

Reverse transcriptase

Answer 10

Reverse transcriptase is an enzyme that converts single-stranded mRNA into double-stranded DNA.

Reverse transcriptase can be used in producing DNA fragments by converting the mRNA for the target gene into double-stranded DNA.

The DNA produced from reverse transcriptase is called complementary DNA (cDNA).

Question 11

E.g. Insulin

Answer 11

DNA fragments of insulin are isolated from pancreatic cells in the following steps:

mRNA for insulin is isolated from the pancreatic cells.

The mRNA is mixed with reverse transcriptase.

Reverse transcriptase converts mRNA into cDNA.

The cDNA can now be used to produce recombinant DNA.

Question 12

Recognition sequences

Answer 12

Recognition sequences are sections of DNA where the base sequence has palindromic base pairs.

Palindromic base pairs have a sequence of base pairs that are the same but in opposite directions.

Recognition sequences can be used to isolate the target gene if there are two sets of sequences either side of the gene.

Question 13

Restriction endonucleases

Answer 13

Enzymes called restriction endonucleases bind to recognition sequences.

Each restriction endonuclease binds to a specific recognition sequence (e.g. Eco RI is a restriction endonuclease that binds to GAATTC).

If two restriction endonucleases bind to two recognition sequences surrounding a target gene, the target gene can be cut out of the DNA.

Question 14

Producing the fragment

Answer 14

DNA fragments can be produced in this way using restriction endonucleases.

The steps involved are -
DNA containing the target gene is mixed with the restriction endonucleases.
Restriction endonucleases bind to the recognition sequences on either side of the target gene.
The target gene is cut out of the DNA.

Question 15

Steps for DNA fragments being produced using gene machines:

Answer 15

Synthesising DNA
Producing the fragment

Question 16

Synthesising DNA

Answer 16

DNA fragments can be produced by synthesising the target gene sequence using free-floating nucleotides.

This is useful because a DNA template isn’t necessary.

The sequence for the target gene is obtained from a database.

Question 17

Producing the fragments

Answer 17

DNA fragments can be produced in this way in the following steps -

The sequence for the target gene is obtained from a database.

Nucleotides are added in the correct order to synthesise the correct base sequence.

Protecting groups are added throughout the synthesis to make sure the correct nucleotides are added and no side branches are produced.

Question 18

The steps involved for in vivo amplification are:

Answer 18

Forming sticky ends
Sticky ends on fragment DNA
Inserting into vector DNA
Transferring to host cells
Inserting marker genes
Identifying transformed cells

Question 19

Forming sticky ends

Answer 19

A vector is a form of transport for the DNA fragment.

Vector DNA is cut open by enzymes called restriction endonucleases.

The enzymes cut the DNA at a specific region called recognition sequences.

Restriction endonucleases cut the vector DNA so that each end has a short single-stranded section.

The ends of the DNA that are single-stranded are called the sticky ends.

Question 20

Sticky ends on fragment DNA

Answer 20

The DNA fragments have sticky ends that are complementary to the sticky ends on the vector DNA.

This is because the DNA fragments have either been cut from DNA using the same restriction endonucleases or because several nucleotides have been added onto the ends of the fragment.

Question 21

Inserting into vector DNA

Answer 21

The sticky ends on the DNA fragment and vector DNA bind together.

An enzyme called DNA ligase attaches the sticky ends together.

This is called ligation.

The DNA fragment has been inserted into the vector DNA.

This is recombinant DNA.

Question 22

Transferring to host cells

Answer 22

The vector transfers the recombinant DNA to the host cells.

If the vector is a plasmid (small, circular DNA found in bacteria) -

The host cells take up the recombinant DNA via heat-shock.

his is where the cells are heated at 42°C for one minute.

If the vector is a bacteriophage (virus) -

The recombinant DNA is injected into host cells.

Question 23

Inserting marker genes

Answer 23

The cells that have successfully taken up the recombinant DNA are transformed.

Transformed cells are also said to be genetically modified (GM).

Not all the cells will be transformed.

The transformed cells are identified using marker genes.

Marker genes are genes that are inserted along with the recombinant DNA and confer antibiotic resistance.

Question 24

Identifying transfromed cells

Answer 24

Transformed cells can be identified by placing the cells on an agar plate with antibiotics.

Only cells that have successfully taken up the recombinant DNA will be able to survive on the antibiotic agar plates.

Transformed cells can then be grown in large numbers to amplify the target gene.

Question 25

The steps involved for in vitro amplification are:

Answer 25

Set up the reaction mixture Heat to 95 degrees Cool to 65 degrees Heat to 72 degrees Repeat

Question 26

Set up the reaction mixture

Answer 26

The DNA fragments are mixed with - Primers (short sections of DNA). An enzyme called DNA polymerase (produces new strands of DNA). Free-floating nucleotides. Together these components form the reaction mixture.

Question 27

Heat to 95°C

Answer 27

Heat the reaction mixture to 95°C. The high heat causes the hydrogen bonds between DNA strands to break and the DNA to separate into two separate strands.

Question 28

Cool to 65°C

Answer 28

Cool the reaction mixture to 65°C. This causes the primer to anneal to the two separate strands of DNA. The primers are complementary to the beginning of the two strands.

Question 29

Heat to 72°C

Answer 29

Heat the reaction mixture to 72°C. This is the optimum temperature for DNA polymerase activity. DNA polymerase produces two new strands of DNA by using the two separated strands of DNA as a template. DNA polymerase adds free-floating nucleotides that are complementary to the template strands of DNA. Primers allow the nucleotides to bind to one another and produce a strand of DNA.

Question 30

Repeat

Answer 30

This process of heating, cooling and heating produces two new strands of DNA from one strand. The process can repeated as many times as possible to quickly amplify the number of DNA fragments. The number of DNA fragments is doubled in each cycle of PCR.

Question 31

Recombinant DNA has a variety of applications. These applications can be hugely beneficial in combating a number of humanitarian issues. These include:

Answer 31

Genetically modified crops Genetically modified livestock Increased nutritional value Treating diseases Industry

Question 32

Genetically modified crops

Answer 32

Recombinant DNA can be used to genetically modify crops to improve their yield. Traits that can be improved include - Resistance to disease. Tolerance to the application of herbicides and pesticides. Tolerance of adverse environmental conditions (e.g. drought).

Question 33

Genetically modified livestock

Answer 33

Recombinant DNA can be used by farmers to make the production of meat more economically viable. Traits that can be improved include - Grow faster and larger. Resistance to disease.

Question 34

Increased nutritional value

Answer 34

Recombinant DNA can be used to increase the nutritional value of food. E.g. Rice has been genetically modified to contain Vitamin A. Vitamin A is a common deficiency in Asian countries where rice is widely consumed.

Question 35

Treating diseases

Answer 35

Recombinant DNA can be used to produce medicine and hormones to treat diseases. E.g. Individuals with type I diabetes used to be given pig insulin to control their blood sugar levels. Now human insulin is created using genetically modified bacteria.

Question 36

Industry

Answer 36

Recombinant DNA can be used to manufacture enzymes. These enzymes can be used in industry. E.g. Rennet is an enzyme traditionally taken from the stomach of young mammals like calves to produce cheese. It is now possible to make rennet using genetically engineered bacteria.

Question 37

Although recombinant DNA technology can be hugely beneficial, there are concerns raised by environmentalists and anti-globalisation activists in opposition to the use of recombinant DNA technology.

Answer 37

Spread of genes Unforseen impacts Economic consequences Medical uses

Question 38

Spread of genes

Answer 38

Genetically modified (GM) crops and livestock are produced when a beneficial gene is inserted into their genome to improve a certain trait. The genes could be transferred into other organisms where it is harmful. E.g. A gene for herbicide resistance could be passed on to a weed, or a gene for antibiotic resistance to pathogenic bacteria. Genes from genetically engineered (transgenic) crops could also spread to organic crops.

Question 39

Unforseen impacts

Answer 39

Genetic modifications to an organism could have unforeseen effects and disrupt normal gene function. The use of genetically engineered organisms could lead to a reduction in the variety in populations. If variety in a population decreases, biodiversity also decreases. Low biodiversity can have negative impacts (e.g. extinction is more likely).

Question 40

Economic consequences

Answer 40

There could be economic consequences for some countries if genetically engineered crops can be grown in different countries where previously it was not possible. Companies who are able to invest more money in recombinant DNA technology may out-compete others.

Question 41

Medical uses

Answer 41

Some activists are concerned that using recombinant DNA in medicine could lead to unethical uses of genetic engineering. E.g. Selecting specific traits in offspring (designer babies).

Question 42

Gene therapy

Answer 42

Gene therapy is a genetic engineering technique used to cure disease.

Question 43

Gene therapy proceure

Answer 43

Gene therapy involves the introduction of a target gene (the gene that confers a beneficial trait) into the genome. The genome has been transformed. The target gene is then transcribed and translated to produce the desired protein. The protein counteracts the effect of a disease that is caused by a mutation.

Question 44

Allele interactions

Answer 44

Gene therapy is used to treat diseases that are caused by a mutation in a gene. The way that gene therapy is used depends on the allele interactions of the gene that causes the disease. If the mutation is in the recessive allele, a wild-type (typical of the species) dominant allele is inserted into the genome. The dominant allele counteracts the mutant alleles. If the mutation is in the dominant allele, an allele that 'silences' the mutant allele is inserted in the genome.

Question 45

Vectors

Answer 45

Gene therapy uses vectors to insert the target gene into the genome. Vectors transport allow the gene to be taken up by the cells of the host. The genome is then transformed. Types of vector include plasmids and bacteriophages.

Question 46

Types of gene therapy

Answer 46

There are two types of gene therapy - Somatic therapy - altering of alleles in adult body cells. Germline therapy - altering of alleles in sex cells. This is illegal in humans.