Appl of reproduction & genetics

Human Genome Project

an international research project involving thousands of scientists which used Sanger sequencing to successfully map the entire human genome

DNA sequencing

identifying the base sequence of a DNA fragment

Sanger sequencing

a method of DNA sequencing that only sequences relatively short sections of DNA at a time, takes a long period of time

Potential benefits of the Human Genome Project

• allows development of targeted, personalised medical treatments + greater accuracy of diagnosis

• increased opportunities for screening genetic conditions + early detection of disease

• enables the study of indices of mutation in different genes

Gene

a length of DNA that codes for a polypeptide

100K Genome Project

UK Government project that aims to study variation in the human genome amongst 100,000 UK citizens, using Next Generation Sequencers

Next Generation Sequencing

a faster, cheaper + more accessible method of sequencing that can sequence an entire genome in a few hours

Genetic counselling

• service that provides information + advice to people affected by or at risk of genetic diseases

• helps individuals + families to make informed decisions

Genetic screening

• testing individuals for certain faulty alleles

• used to detect disorders such as cystic fibrosis, Huntington’s and thalassemia

Potential disadvantages of genetic screening

• screening for conditions like cancer/Alzheimer’s only indicates increased risk - may cause unnecessary worry

• test result data may be used in discrimination from employers/insurance companies, may be misused

• risk of false positives/negatives

• who should be screened? Limited funds + time

• screening embryos could lead to ‘designer babies’

Examples of non-human organisms whose genomes have been sequences

Chimpanzees, the mosquito, Plasmodium parasite

How has sequencing the genome of the mosquito been useful to humans

• Anopheles has developed insecticide resistance

• sequencing has enabled the development of chemicals that make Anopheles susceptible to insecticides

Advantage of sequencing the genome of Plasmodium parasite

plasmodium has developed multi-drug resistance, enables the development of more effective drugs

Genetic fingerprinting

a technique used to genetically identify an organism

Uses of genetic profiling/fingerprinting

• paternity testing

• determining twins type

• finding siblings

• for immigration

• forensic use

• phylogenetic studies

Exons

regions of DNA that code for polypeptides

Introns

non-coding sequences of DNA

What is satellites and stutter sequences another name for

introns

STRs

Short Tandem Repeats, sections of repeated nucleotides within introns that produce variation in individuals

DNA amplification

increasing the amount of DNA

What techniques can be used to produce a genetic fingerprint

PCR + gel electrophoresis

PCR

Polymerase Chain Reaction, an in vitro technique used to rapidly amplify fragments of DNA

Describe the reaction mixture in the first stage of PCR

• DNA fragment to be amplified

• primers complimentary to start of fragment

• free nucleotides to match exposed bases

• Taq DNA polymerase to create new DNA

Taq DNA polymerase

a thermally stable enzyme that synthesises a double-stranded molecule of DNA from a single template strand using complementary nucleotides

Primers

pieces of single-stranded DNA which are complementary to the specific target sequence at the 3’ end of each DNA replicated strand to be replicated

Process of amplifying DNA fragments using PCR

1. heated to 95 degrees to break hydrogen bonds between DNA strands

2. cooled to 60 degrees to allow primers to bind - annealing

3. heated to 72 degrees to activate Taq DNA polymerase + allow free nucleotides to join

4. new DNA acts as a template for next cycle

Gel electrophoresis

a technique that separates nucleic acid fragments/proteins by size using electric current

How does gel electrophoresis work

• DNA fragments of varying lengths are placed at 1 end of a slab or agarose gel

• electric current applied, DNA fragments move towards the positive end of the gel

• shorter fragments travel further. The pattern of bands created is unique to every individual

Genetic engineering

modification of the genome of an organism by the insertion of a desired gene from another organism. Enables the formation of organisms with beneficial characteristics

Recombinant DNA

a combination of DNA from 2 different organisms

Process of using restriction enzymes to produce DNA fragments

• gene identified using gene probe

• restriction endonucleases cut DNA at specific palindromic sequences producing sticky ends

Process of using reverse transcriptase to produce DNA fragments

• mRNA complementary to target gene used as a template

• reverse transcriptase synthesises cDNA

• mixed with free nucleotides which match up to their base pairs

• DNA polymerase joins nucleotides forming 2nd strand

Advantages of using reverse transcriptase to produce cDNA

• don’t have to locate the gene

• gene not cut into non-functional fragments by restriction enzymes

• introns not present in cDNA

• doesn’t require post-transcriptional processing to produce functional mRNA

Process of inserting a DNA fragment into a vector

• plasmid used as vector

• plasmid cut using same restriction enzymes as DNA, so sticky ends are complementary

• DNA ligase joins fragment + plasmid together

How are antibiotic-resistant genes are used in identifying recombinant bacteria

antibiotic resistance genes can be inserted into plasmids at the same time as DNA fragments. The transformed cells are then placed on a plate with antibiotics. Only the cells that successfully took up the vector will grow

Give an application for genetic modification of bacterial cells

human gene for insulin production can be inserted into a vector, so that the bacterial cell will produce insulin. Useful in medicine e.g. treatment of diabetes

Disadvantages of using recombinant DNA to make human products

• identifying the required gene may be difficult

• some eukaryotic genes can’t be expressed in prokaryotes

• antibiotic-resistance genes can be transferred into pathogenic bacteria

• expensive

GM organisms

organisms that have had their genome altered

Benefits of GM crop production

• improves nutritional value of foods

• longer shelf-life of products

• greater crop yields & reduces crop losses

• reduces need for land clearing

• can produce human medicine & vaccines

Risks of GM production

• reduction in biodiversity

• unknown effects on health

• cross-pollination could result in herbicide-resistant weeds

• may increase costs for farmers

• insect eating predators may be damaged by toxins in plant

• transferred gene may spread to wild population and cause problems

Gene therapy

a therapeutic technique in which a faulty allele is replaced with a functional allele in order to treat/prevent disease

2 types of gene therapy

somatic cell therapy and germ line therapy

Somatic cell therapy

allele introduced to target cells only, short-term, must be repeated

Germ line therapy

allele introduced to embryonic cells so it is present in all resultant cells, permanent, passed onto offspring

Vector

a carrier used to transfer a gene from one organism to another e.g. plasmid or virus

Duchenne Muscular Dystrophy

X-linked recessive condition, characterised by muscle degeneration and weakness

Cause of DMD

1 or more mutations in the dystrophin gene that prevent the production of dystrophin

How can DMD be treated using gene therapy

1. healthy gene inserted into vector

2. vector inserted into muscle tissue

3. virus delivers gene to muscle cells

4. new gene incorporated into DNA of cell

5. transcription and translation of gene produces normal dystrophin protein

6. symptoms of DMD alleviated

Drisapersen

an experimental drug that aims to treat DMD by exon skipping

How does drisapersen work

it introduces a ‘molecular patch’ over the mutated exon, enabling the gene to be read. A shorter, more functional type of dystrophin is synthesised

Ethical issues surrounding gene therapy

• health implications - may produce an immune response, activation of oncogenes

• is it right to alter the genotype of an unborn child?

• what conditions should be treated using gene therapy?

• could lead to healthcare inequalities

• expensive - money could be spent elsewhere

Stem cells

cells that are unspecialised + retain the ability to differentiate into a range of cell types

Tissue engineering

an extension of gene therapy that aims to replace, repair or improve biological function by replacing organs and tissues

Main advantage of using stem cells

rapid production of genetically identical cells and organisms

Disadvantages of using stem cells

• expensive + unreliable in mammals

• disease + pathogens can cause issues in plants

• inadvertent selection of disadvantageous alleles, unknown long-term effects

Ethical issues related to stem cell use from embryos

• embryos used to provide stem cells are destroyed which is seen as unethical and a waste of potential human life

• could lead to the ‘farming’ of embryos for stem cells

• may lead to the reproductive cloning of humans