Amplification

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

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

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

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.

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

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

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.

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

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

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

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

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.

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.

1Biological Molecules

1.1Monomers & Polymers

1.1.1Monomers & Polymers

1.1.2Condensation & Hydrolysis Reactions

1.2Carbohydrates

1.2.1Structure of Carbohydrates

1.2.2Types of Polysaccharides

1.2.3End of Topic Test - Monomers, Polymers and Carbs

1.2.4Exam-Style Question - Carbohydrates

1.2.5A-A* (AO3/4) - Carbohydrates

1.3Lipids

1.3.1Triglycerides & Phospholipids

1.3.2Types of Fatty Acids

1.3.3Testing for Lipids

1.3.4Exam-Style Question - Fats

1.3.5A-A* (AO3/4) - Lipids

1.4Proteins

1.4.1The Peptide Chain

1.4.2Investigating Proteins

1.4.3Primary & Secondary Protein Structure

1.4.4Tertiary & Quaternary Protein Structure

1.4.5Enzymes

1.4.6Factors Affecting Enzyme Activity

1.4.7Enzyme-Controlled Reactions

1.4.8End of Topic Test - Lipids & Proteins

1.4.9A-A* (AO3/4) - Enzymes

1.4.10A-A* (AO3/4) - Proteins

1.5Nucleic Acids

1.5.1DNA & RNA

1.5.2Polynucleotides

1.5.3DNA Replication

1.5.4Exam-Style Question - Nucleic Acids

1.5.5A-A* (AO3/4) - Nucleic Acids

1.6ATP

1.6.1Structure of ATP

1.6.2End of Topic Test - Nucleic Acids & ATP

1.7Water

1.7.1Structure & Function of Water

1.7.2A-A* (AO3/4) - Water

1.8Inorganic Ions

1.8.1Inorganic Ions

1.8.2End of Topic Test - Water & Inorganic Ions

2Cells

2.1Cell Structure

2.1.1Introduction to Cells

2.1.2Eukaryotic Cells & Organelles

2.1.3Eukaryotic Cells & Organelles 2

2.1.4Prokaryotes

2.1.5A-A* (AO3/4) - Organelles

2.1.6Methods of Studying Cells

2.1.7Microscopes

2.1.8End of Topic Test - Cell Structure

2.1.9Exam-Style Question - Cells

2.1.10A-A* (AO3/4) - Cells

2.2Mitosis & Cancer

2.2.1Mitosis

2.2.2Investigating Mitosis

2.2.3Cancer

2.2.4A-A* (AO3/4) - The Cell Cycle

2.3Transport Across Cell Membrane

2.3.1Cell Membrane Structure

2.3.2A-A* (AO3/4) - Membrane Structure

2.3.3Diffusion

2.3.4Osmosis

2.3.5Active Transport

2.3.6End of Topic Test - Mitosis, Cancer & Transport

2.3.7Exam-Style Question - Membranes

2.3.8A-A* (AO3/4) - Membranes & Transport

2.3.9A-A*- Mitosis, Cancer & Transport

2.4Cell Recognition & the Immune System

2.4.1Immune System

2.4.2The Immune Response

2.4.3Antibodies

2.4.4Primary & Secondary Response

2.4.5Vaccines

2.4.6HIV

2.4.7Ethical Issues

2.4.8End of Topic Test - Immune System

2.4.9Exam-Style Question - Immune System

2.4.10A-A* (AO3/4) - Immune System

3Substance Exchange

3.1Surface Area to Volume Ratio

3.1.1Size & Surface Area

3.1.2A-A* (AO3/4) - Cell Size

3.2Gas Exchange

3.2.1Single-Celled Organisms

3.2.2Multicellular Organisms

3.2.3Control of Water Loss

3.2.4Human Gas Exchange

3.2.5Ventilation

3.2.6Dissection

3.2.7Measuring Gas Exchange

3.2.8Lung Disease

3.2.9Lung Disease Data

3.2.10End of Topic Test - Gas Exchange

3.2.11A-A* (AO3/4) - Gas Exchange

3.3Digestion & Absorption

3.3.1Overview of Digestion

3.3.2Digestion in Mammals

3.3.3Absorption

3.3.4End of Topic Test - Substance Exchange & Digestion

3.3.5A-A* (AO3/4) - Substance Ex & Digestion

3.4Mass Transport

3.4.1Haemoglobin

3.4.2Oxygen Transport

3.4.3The Circulatory System

3.4.4The Heart

3.4.5Blood Vessels

3.4.6Cardiovascular Disease

3.4.7Heart Dissection

3.4.8Xylem

3.4.9Phloem

3.4.10Investigating Plant Transport

3.4.11End of Topic Test - Mass Transport

3.4.12A-A* (AO3/4) - Mass Transport

4Genetic Information & Variation

4.1DNA, Genes & Chromosomes

4.1.1DNA

4.1.2Genes

4.1.3A-A* (AO3/4) - DNA

4.2DNA & Protein Synthesis

4.2.1Protein Synthesis

4.2.2Transcription & Translation

4.2.3End of Topic Test - DNA, Genes & Protein Synthesis

4.2.4Exam-Style Question - Protein Synthesis

4.2.5A-A* (AO3/4) - Coronavirus Translation

4.2.6A-A* (AO3/4) - Transcription

4.2.7A-A* (AO3/4) - Translation

4.3Mutations & Meiosis

4.3.1Mutations

4.3.2Meiosis

4.3.3A-A* (AO3/4) - Meiosis

4.3.4Meiosis vs Mitosis

4.3.5End of Topic Test - Mutations, Meiosis

4.3.6A-A* (AO3/4) - DNA,Genes, CellDiv & ProtSynth

4.4Genetic Diversity & Adaptation

4.4.1Genetic Diversity

4.4.2Natural Selection

4.4.3A-A* (AO3/4) - Natural Selection

4.4.4Adaptations

4.4.5Investigating Natural Selection

4.4.6End of Topic Test - Genetic Diversity & Adaptation

4.4.7A-A* (AO3/4) - Genetic Diversity & Adaptation

4.5Species & Taxonomy

4.5.1Classification

4.5.2DNA Technology

4.5.3A-A* (AO3/4) - Species & Taxonomy

4.6Biodiversity Within a Community

4.6.1Biodiversity

4.6.2Agriculture

4.6.3End of Topic Test - Species,Taxonomy& Biodiversity

4.6.4A-A* (AO3/4) - Species,Taxon&Biodiversity

4.7Investigating Diversity

4.7.1Genetic Diversity

4.7.2Quantitative Investigation

5Energy Transfers (A2 only)

6Responding to Change (A2 only)

6.1Nervous Communication

6.1.1Survival

6.1.2Plant Responses

6.1.3Animal Responses

6.1.4Reflexes

6.1.5End of Topic Test - Reflexes, Responses & Survival

6.1.6Receptors

6.1.7The Human Retina

6.1.8Control of Heart Rate

6.1.9End of Topic Test - Receptors, Retina & Heart Rate

6.2Nervous Coordination

6.2.1Neurones

6.2.2Action Potentials

6.2.3Speed of Transmission

6.2.4End of Topic Test - Neurones & Action Potentials

6.2.5Synapses

6.2.6Types of Synapse

6.2.7Medical Application

6.2.8End of Topic Test - Synapses

6.2.9A-A* (AO3/4) - Nervous Comm&Coord

6.3Muscle Contraction

6.3.1Skeletal Muscle

6.3.2Sliding Filament Theory

6.3.3Contraction

6.3.4Slow & Fast Twitch Fibres

6.3.5End of Topic Test - Muscles

6.3.6A-A* (AO3/4) - Muscle Contraction

6.4Homeostasis

6.4.1Overview of Homeostasis

6.4.2Blood Glucose Concentration

6.4.3Controlling Blood Glucose Concentration

6.4.4End of Topic Test - Blood Glucose

6.4.5Primary & Secondary Messengers

6.4.6Diabetes Mellitus

6.4.7Measuring Glucose Concentration

6.4.8Osmoregulation

6.4.9Controlling Blood Water Potential

6.4.10ADH

6.4.11End of Topic Test - Diabetes & Osmoregulation

6.4.12A-A* (AO3/4) - Homeostasis

7Genetics & Ecosystems (A2 only)

7.1Genetics

7.1.1Key Terms in Genetics

7.1.2Inheritance

7.1.3Linkage

7.1.4Multiple Alleles & Epistasis

7.1.5Chi-Squared Test

7.1.6End of Topic Test - Genetics

7.1.7A-A* (AO3/4) - Genetics

7.2Populations

7.2.1Populations

7.2.2Hardy-Weinberg Principle

7.3Evolution

7.3.1Variation

7.3.2Natural Selection & Evolution

7.3.3End of Topic Test - Populations & Evolution

7.3.4Types of Selection

7.3.5Types of Selection Summary

7.3.6Overview of Speciation

7.3.7Causes of Speciation

7.3.8Diversity

7.3.9End of Topic Test - Selection & Speciation

7.3.10A-A* (AO3/4) - Populations & Evolution

7.4Populations in Ecosystems

7.4.1Overview of Ecosystems

7.4.2Niche

7.4.3Population Size

7.4.4Investigating Population Size

7.4.5End of Topic Test - Ecosystems & Population Size

7.4.6Succession

7.4.7Conservation

7.4.8End of Topic Test - Succession & Conservation

7.4.9A-A* (AO3/4) - Ecosystems

8The Control of Gene Expression (A2 only)

8.1Mutation

8.1.1Mutations

8.1.2Effects of Mutations

8.1.3Causes of Mutations

8.2Gene Expression

8.2.1Stem Cells

8.2.2Stem Cells in Disease

8.2.3End of Topic Test - Mutation & Gene Epression

8.2.4A-A* (AO3/4) - Mutation & Stem Cells

8.2.5Regulating Transcription

8.2.6Epigenetics

8.2.7Epigenetics & Disease

8.2.8Regulating Translation

8.2.9Experimental Data

8.2.10End of Topic Test - Transcription & Translation

8.2.11Tumours

8.2.12Correlations & Causes

8.2.13Prevention & Treatment

8.2.14End of Topic Test - Cancer

8.2.15A-A* (AO3/4) - Gene Expression & Cancer

8.3Genome Projects

8.3.1Using Genome Projects

8.4Gene Technology

8.4.1Recombinant DNA

8.4.2Producing Fragments

8.4.3Amplification

8.4.4End of Topic Test - Genome Project & Amplification

8.4.5Using Recombinant DNA

8.4.6Medical Diagnosis

8.4.7Genetic Fingerprinting

8.4.8End of Topic Test - Gene Technologies

8.4.9A-A* (AO3/4) - Gene Technology

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