19.1.2
Restriction Endonucleases
Restriction Endonucleases
Restriction Endonucleases
The target gene for producing recombinant DNA is obtained by producing DNA fragments. DNA fragments can be produced using restriction endonuclease enzymes.
Recognition sequences
Recognition sequences
- Recognition sequences are sections of DNA where the base sequence has antiparallel base pairs.
- Antiparallel 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.
Restriction endonucleases
Restriction endonucleases
- 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.
Producing the fragment
Producing the fragment
- 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.
Reverse transcriptase
Reverse transcriptase
- Rather than cutting out the gene with restriction endonucleases, reverse transcriptase can be used in an alternative method.
- Reverse transcriptase can convert the mRNA of the desired gene into DNA.
- The mRNA can be extracted from target cells.
In vivo
In vivo
After DNA fragments have been produced, they can be amplified either in vivo (inside the organism) or in vitro (outside the organism). The steps involved for in vivo amplification are:
1) Forming sticky ends
1) Forming sticky ends
- 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.
2) Sticky ends on fragment DNA
2) Sticky ends on fragment DNA
- 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.
3) Inserting into vector DNA
3) Inserting into vector DNA
- 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.
- The DNA fragment needs to contain promoter sequences to ensure transcription.
4) Transferring to host cells
4) Transferring to host cells
- 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. This 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.
5) Inserting marker genes
5) Inserting marker genes
- 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.
6) Identifying transformed cells
6) Identifying transformed cells
- 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.
- Sometimes fluorescent genes or genes for easily dyed substances are also used as marker genes.
In vitro
In vitro
In vitro amplification uses the polymerase chain reaction (PCR). PCR can rapidly increase the number of copies of DNA fragments. The steps involved for in vitro amplification are:
1) Set up the reaction mixture
1) Set up the reaction mixture
- 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.
2) Heat to 95°C
2) Heat to 95°C
- 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.
3) Cool to 65°C
3) Cool to 65°C
- 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.
4) Heat to 72°C
4) Heat to 72°C
- Heat the reaction mixture to 72°C.
- This is the optimum temperature for Taq polymerase activity.
- Taq polymerase is a DNA polymerase enzyme from organisms that grow at high temperatures.
- Taq polymerase produces two new strands of DNA by using the two separated strands of DNA as a template.
- Taq 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.
5) Repeat
5) Repeat
- 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.
1Cell Structure
1.1Cell Structure
1.1.1Studying Cells - Microscopes
1.1.2Introduction to Eukaryotic & Prokaryotic Cells
1.1.3Ultrastructure of Eukaryotic Cells
1.1.4Ultrastructure of Eukaryotic Cells 2
1.1.5Ultrastructure of Eukaryotic Cells 3
1.1.6Prokaryotic Cells
1.1.7Viruses
1.1.8End of Topic Test - Cell Structure
1.1.9Exam-Style Question - Microscopes
1.1.10A-A* (AO2/3) - Cell Structure
2Biological Molecules
2.1Testing for Biological Modules
2.2Carbohydrates & Lipids
2.3Proteins
3Enzymes
4Cell Membranes & Transport
4.1Biological Membranes
5The Mitotic Cell Cycle
6Nucleic Acids & Protein Synthesis
6.1Nucleic Acids
7Transport in Plants
8Transport in Mammals
8.1Circulatory System
8.2Transport of Oxygen & Carbon Dioxide
9Gas Exchange
9.1Gas Exchange System
10Infectious Diseases
10.1Infectious Diseases
10.2Antibiotics
11Immunity
12Energy & Respiration (A2 Only)
13Photosynthesis (A2 Only)
14Homeostasis (A2 Only)
14.1Homeostasis
14.2The Kidney
14.3Cell Signalling
14.4Blood Glucose Concentration
14.5Homeostasis in Plants
15Control & Coordination (A2 Only)
15.1Control & Coordination in Mammals
15.1.1Neurones
15.1.2Receptors
15.1.3Taste
15.1.4Reflexes
15.1.5Action Potentials
15.1.6Saltatory Conduction
15.1.7Synapses
15.1.8Cholinergic Synnapses
15.1.9Neuromuscular Junction
15.1.10Skeletal Muscle
15.1.11Sliding Filament Theory Contraction
15.1.12Sliding Filament Theory Contraction 2
15.1.13Menstruation
15.1.14Contraceptive Pill
15.2Control & Co-Ordination in Plants
16Inherited Change (A2 Only)
16.1Passage of Information to Offspring
16.2Genes & Phenotype
17Selection & Evolution (A2 Only)
17.2Natural & Artificial Selection
18Classification & Conservation (A2 Only)
18.1Biodiversity
18.2Classification
19Genetic Technology (A2 Only)
19.1Manipulating Genomes
19.2Genetic Technology Applied to Medicine
19.3Genetically Modified Organisms in Agriculture
Jump to other topics
1Cell Structure
1.1Cell Structure
1.1.1Studying Cells - Microscopes
1.1.2Introduction to Eukaryotic & Prokaryotic Cells
1.1.3Ultrastructure of Eukaryotic Cells
1.1.4Ultrastructure of Eukaryotic Cells 2
1.1.5Ultrastructure of Eukaryotic Cells 3
1.1.6Prokaryotic Cells
1.1.7Viruses
1.1.8End of Topic Test - Cell Structure
1.1.9Exam-Style Question - Microscopes
1.1.10A-A* (AO2/3) - Cell Structure
2Biological Molecules
2.1Testing for Biological Modules
2.2Carbohydrates & Lipids
2.3Proteins
3Enzymes
4Cell Membranes & Transport
4.1Biological Membranes
5The Mitotic Cell Cycle
6Nucleic Acids & Protein Synthesis
6.1Nucleic Acids
7Transport in Plants
8Transport in Mammals
8.1Circulatory System
8.2Transport of Oxygen & Carbon Dioxide
9Gas Exchange
9.1Gas Exchange System
10Infectious Diseases
10.1Infectious Diseases
10.2Antibiotics
11Immunity
12Energy & Respiration (A2 Only)
13Photosynthesis (A2 Only)
14Homeostasis (A2 Only)
14.1Homeostasis
14.2The Kidney
14.3Cell Signalling
14.4Blood Glucose Concentration
14.5Homeostasis in Plants
15Control & Coordination (A2 Only)
15.1Control & Coordination in Mammals
15.1.1Neurones
15.1.2Receptors
15.1.3Taste
15.1.4Reflexes
15.1.5Action Potentials
15.1.6Saltatory Conduction
15.1.7Synapses
15.1.8Cholinergic Synnapses
15.1.9Neuromuscular Junction
15.1.10Skeletal Muscle
15.1.11Sliding Filament Theory Contraction
15.1.12Sliding Filament Theory Contraction 2
15.1.13Menstruation
15.1.14Contraceptive Pill
15.2Control & Co-Ordination in Plants
16Inherited Change (A2 Only)
16.1Passage of Information to Offspring
16.2Genes & Phenotype
17Selection & Evolution (A2 Only)
17.2Natural & Artificial Selection
18Classification & Conservation (A2 Only)
18.1Biodiversity
18.2Classification
19Genetic Technology (A2 Only)
19.1Manipulating Genomes
19.2Genetic Technology Applied to Medicine
19.3Genetically Modified Organisms in Agriculture
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