Eduqas Biology A Level C2 - Application of reproduction and genetics

What are the aims of the Human Genome Project?

- To identify all the genes in the human genome and which chromosome each of them are on

- To determine the sequence of the bases in human DNA and store the information in databases

- Improve tools for data analysis

- Improve medical knowledge on genetic diseases

- Address the ethical, legal and social issues that may arise from the project

What method of sequencing was used by the Human Genome Project?

Sanger sequencing

Define electrophoresis

A lab technique that separates molecules on the basis of size, by their rate of migration under an applied voltage

What are the downsides of Sanger sequencing?

It takes a very long time to sequence the human genome (1 year to sequence 1 million bases)

What are the aims of the 100K Genome Project?

- Create an ethical, transparent programme based on consent

- Set up a genomic service for the NHS to benefit patients

- Enable medical and scientific discovery

- Develop a UK genomics indusry

- Identify causes of genetic diseases

- Develop better treatments for genetic diseases

What are the ethical issues tackled that concern genome sequencing?

- Ownership of genetic information

- The identification of future genetic diseases, which some people may not want to know

- Genetic counselling and the benefit DNA sequencing has on it

- Embryos can be screened for genetic diseases, and may be aborted (or not used during IVF)

- The potential for choosing alleles to ensure specific characteristics in embryos

- Parents may want to screen their children, so a decision has to be made as to whether to tell them

- Storage and security of genetic data

What is an example of genetic modification in an organism other than humans?

Targeting malaria by killing the parasite and vector

How has the use of DNA sequencing aided the killing of mosquitoes?

DNA sequencing of the mosquito has enabled the development of chemicals that make them susceptible to insecticides (as insecticide resistance is a large problem)

Genetically modified infertile mosquitoes have also been developed, aiding research

What is the scientific name of the mosquito?

Anopheles gambiae

What is the genus of the malaria parasite?

Plasmodium

How has the use of DNA sequencing aided the killing of the malaria parasite Plasmodium?

It is allowing for the development of more effective drugs

Genetic fingerprinting relies on what two techniques?

PCR and gel electrophoresis

What does STR stand for?

short tandem repeats

What is a short tandem repeat (STR)?

Sequences of nucleotides in introns in which up to 13 bases repeat up to several hundred times

What does PCR stand for?

polymerase chain reaction

What is a primer?

A strand of DNA about 10 nucleotides long that base pairs with the end of another longer strand, making a double-stranded section, to which DNA polymerase can attach prior to replication

What is the purpose of PCR?

To amplify DNA (make lots of copies of DNA)

During PCR, what 3 things must be mixed with the target DNA?

- Taq polymerase

- DNA nucleotides

- Primers

Describe the process of PCR

- The solution containing the target DNA is heated to 95°C

- The solution is cooled to 50°C

- The solution is then heated to 70°C

- The sequence is repeated many times

Why is the solution first heated to 95°C in PCR?

The heat breaks the hydrogen bonds between the DNA strands, separating them into two single strands

Why is the solution cooled to 50°C in PCR after being heated to 95°C?

This allows the primers to anneal to the complementary base sequences on each of the single strands of DNA

Why is the solution heated to 70°C in PCR after being heated to 95°C then cooled to 50°C?

This is the optimum temperature for the enzyme Taq polymerase, which catalyses the synthesis of s complimentary stand

What are the limitations of PCR?

- Contamination of samples with non-target DNA

- Error rate (increases the more repeats you do)

- PCR is most efficient for fragments of DNA between 1000 and 4000 base pairs, but human genes are much longer

- PCR is very sensitive to inhibitors

- After about 20 cycles the rate of replication slows because reagent concentrations become limiting, and the enzyme is denatured after multiple heating

Describe the process of gel electrophoresis

- DNA is extracted and cut into fragments by restriction endonucleases

- The sample is applied to wells in agarose gel

- A voltage is applied across the gel, causing the fragments to move to the positive end

- Smaller fragments move further along the gel

- Southern blotting and X-rays are used to develop a more clear pattern of dark bands

What are restriction endonucleases?

Restriction enzymes, enzymes that cut DNA at specific nucleotide sequences

What is the purpose of a DNA template ladder?

It contains fragments of known length, so allows you to see how many base pairs each fragment has

How do you extract DNA from a cell?

Using detergents, which break open the cell membrane and nuclear envelope

What are some uses of DNA profiling?

- Paternity tests

- Testing for identical twins

- Testing for blood relation between siblings

- Some visa applications rely on proof of relatedness

- Forensic use (for determining suspects)

-Phylogenetic studies

What are the pros of DNA profiling?

- Biological samples can be obtained by non-invasive methods, such as cheek swabs, rather than blood samples

- Can be done on samples too small for blood testing

- Has reversed wrong convictions in criminal trials

- It can be used to exonerate people who have been falsely accused

What are the cons of DNA profiling?

- Some consider a request for a DNA sample is a violation of an individual's privacy

- DNA profiles held in computer databases are vulnerable to cyber attack

- DNA profiles offer probabilities, not absolutes

- Access to and use of data must be carefully regulated (could be used to deny insurance coverage or claims)

- DNA profiling may produce wrongful convictions if it is used irresponsibly

Define recombinant DNA

DNA produced by combining DNA from two different species

Define transgenic

An organism that has been genetically modified by the addition of a gene or genes from another species

Summarise the process of producing a protein using genetic engineering

- DNA fragment is isolated

- The DNA fragment is inserted into a vector

- The vector is then inserted into a suitable host cell

- Gene markers are used to confirm the gene has been taken up by the host cell

- The host cell is cloned

What two methods are used to isolate a gene during genetic engineering?

Using restriction endonuclease or using reverse transcriptase

Define sticky end

A sequence of unpaired bases on a double-stranded DNA molecule that readily base pairs with a complimentary strand

Describe the process of isolating a gene using restriction endonuclease

- A restriction endonuclease is used to cut DNA at a specific site, making fragments

- The restriction endonuclease makes a jagged cut, leaving sticky ends on the DNA fragment

- This fragment can now be inserted into a vector

What are the two main drawbacks of using restriction endonucleases?

- If the specific nucleotide sequence the restriction endonuclease cuts occurs within the gene, the gene will be broken into fragments

- Introns will be present, and bacteria may not be able to process the mRNA made from the vector if they are present

What is reverse transcriptase?

An enzyme that catalyses the synthesis of cDNA from an RNA molecule

What does the "c" in cDNA stand for?

complementary

Describe the process of gene isolation using reverse transcriptase

- A specific mRNA molecule is extracted

- The enzyme reverse transcriptase synthesises a DNA stand from the mRNA molecule, which is complimentary to the mRNA and called cDNA

- DNA polymerase then catalyses the synthesis of a DNA molecule from the single cDNA strand

Define vector

A virus or plasmid used as a vehicle for carrying foreign genetic material into a cell

Define plasmid

A small circular loop of self-replicating double-stranded DNA in bacteria

What is the enzyme that is used to permanently join a DNA fragment to a plasmid?

DNA ligase

What enzyme is used to cut open a plasmid?

Restriction endonucleases

What is a marker gene?

A gene that indicates the presence of a plasmid

What are genetic markers used for?

Genetic markers are important for testing whether or not a piece of recombinant DNA has been taken up by the host cell

What are some examples of marker genes?

antibiotic resistance and fluorescent genes

Pros of genetic engineering in bacteria

- Medical products such as insulin can be created more readily

- Tooth decay: modified strains of bacteria that do not cause tooth decay out-compete the decay causing bacteria

- Prevention and treatment of diseases (can produce vaccines)

- Enhancing crop growth

- Environmental use (detecting and removing hazards)

Cons of genetic engineering in bacteria

- Plasmids are easily transferred, so may be transferred into non-target cells and cause antibiotic-resistance

- Fragments of DNA used may contain oncogenes (cancer causing)

- A micro-organism with a new gene may become a threat if released into the environment

- A newly-introduced gene may disrupt the function of another

What are some benefits of GM crops?

- Higher crop yields

- Superior keeping qualities

- Pesticide reduction

- Improved nutritional quality of food

- Resistance to herbicides

- 'Pharming' (GM plants that produce molecules such as antibodies or hormones)

What are some concerns of GM crops?

- Dispersal of pollen from GM crops, fertilising wild crops

- Pest resistance

- Marker genes containing antibiotic resistance

- Reduction in biodiversity

- New proteins that may have long-term health effects

- Pollen from GM crops could compromise 'organic' crops

What are the concerns around genetic screening?

- Many believe it is an invasion of privacy

- Defective alleles in prenatal tests may lead to more abortions (some consider abortions unethical)

- Individuals with defects may be placed in a high-risk group for insurance purposes to cover their treatment, so their insurance cover would be very expensive

What are the main uses of genetic testing?

- Carrier screening to see if an unaffected person may carry a recessive allele for a disease

- Pre-implantation testing to screen embryos in IVF

- Pre-natal diagnostic testing

- Newborn baby screening

- Testing for adult-onset disorders

- Confirmation that an individual has a suspected disease

- Forensic and identity testing

There are some commercially available gene tests that give a probability of developing a few conditions. What are some limitations of commercialised gene tests?

- They are not regulated or verified

- Only test a small number of genes in the human genome

- It is difficult to interpret a positive result (some people who carry a gene may never develop the disease)

- Laboratory errors may occur

- These tests may provoke anxiety

Two main approaches of gene therapy are...

somatic cell therapy and germ-line therapy

What is somatic cell therapy?

Gene therapy that targets the body cells in the affected tissues. Changes do not pass on to replicated cells, and do not pass on to offspring of the patient

What is germ-line therapy?

Gene therapy that targets germ-line cells such as the oocyte (egg cell). Genetic correction is inherited

What is Duchenne Muscular Dystrophy (DMD)?

A disorder that results in progressive weakening and loss of skeletal muscle

What is Duchenne Muscular Dystrophy (DMD) caused by?

A mutation in the dystrophin gene resulting in the failure to produce dystrophin, which is an important structural component of muscle tissue

Treatments for Duchenne Muscular Dystrophy (DMD) include:

- Gene therapy

- Drugs

What is cystic fibrosis?

It is a genetic disorder of the cell membranes. Causes thick, sticky mucus to build up in air passages and pancreas.

What is a liposome?

A hollow phospholipid sphere used as a vehicle to carry molecules into a cell

Disadvantages of gene therapy

- Only a small proportion of the introduce genes are expressed

- There may be an immune response in the patient