Application of reproduction and genetics: cram notes

• The Human Genome Project (1990 to 2003) found the sequence of the 3 billion base pairs of human DNA and the position of each gene on its chromosome.
• It found about 20 000 genes; less than 2% of the genome codes for protein.
• Aims included improving the diagnosis and treatment of genetic disorders and addressing the ethical issues.
• The 100K Genome Project sequenced the genomes of NHS patients with rare diseases or cancer, and their families, and linked them to medical records.

1. Coloured chain terminator bases stop copying at every possible length.
2. Electrophoresis sorts the fragments by length; the colours read from shortest to longest give the sequence.
3. Next generation sequencing reads millions of fragments in parallel, so it is far faster and cheaper.

• Ownership of genetic information and consent to its use.
• Insurers or employers could discriminate against people with a genetic risk.
• Results affect relatives, who may not want to know.
• Some people prefer not to know about an untreatable condition.
• Genomic data could be hacked or misused.

• Pre-implantation genetic diagnosis tests a cell from an IVF embryo, so only unaffected embryos are implanted.
• Prenatal tests: chorionic villus sampling, amniocentesis, or foetal DNA in the mother's blood copied by PCR.
• Cystic fibrosis and thalassaemia are autosomal recessive; Huntington's disease is autosomal dominant.
• Carrier screening finds people with one recessive allele.

• For: prevents severe disease and allows informed choice.
• Against: embryos are destroyed, selective abortion may increase, false results are possible, and some fear selection for non-medical traits.

• The genomes of Anopheles gambiae (the mosquito vector) and Plasmodium falciparum (the parasite) were both sequenced in 2002.
• Both are becoming resistant: mosquitoes to insecticides and Plasmodium to drugs.
• Uses: make mosquitoes susceptible to insecticides again or less fertile, stop them carrying Plasmodium, and find targets for new drugs and vaccines.
• Comparing genomes shows how closely species are related.

1. 95 °C: hydrogen bonds between bases break and the DNA becomes single stranded.
2. About 55 °C: primers (short single strands of DNA) anneal to the complementary sequence at the start of each strand.
3. About 70 °C: Taq polymerase adds complementary nucleotides to each primer.
4. Each cycle doubles the DNA: copies = 2ⁿ after n cycles from one molecule.

• The plateau happens because primers and nucleotides run out, Taq polymerase denatures after repeated heating, and product strands pair with each other.
• Contaminating DNA is copied too.
• Taq polymerase cannot proofread, so errors are copied.
• It works only for fragments up to a few thousand base pairs, and inhibitors in the sample can stop it.

1. Samples go into wells at the negative electrode.
2. DNA is negatively charged (phosphate groups), so it moves to the positive electrode.
3. Small fragments pass through the pores of the agarose more easily and travel furthest.
4. A DNA ladder of known lengths lets you estimate the size of each band.

1. Primers bind either side of the repeats, so PCR copies the whole repeat region.
2. More repeats give a longer fragment, which moves less far on the gel.

Making a genetic fingerprint

1. 1) Extract DNA
2. 2) Copy STRs by PCR
3. 3) Separate by electrophoresis
4. 4) Compare the band patterns

1. Match the child's bands to the mother first.
2. The remaining bands must come from the father; the man who has all of them is very probably the father.

• Uses: forensics, paternity and immigration, twins, conservation such as tracing ivory, and comparing species.
• For: tiny non-invasive samples, and it can clear the innocent.
• Against: privacy, database security, and a match is a probability not a certainty.

1. Restriction enzymes cut DNA at a specific base sequence, often a palindrome such as GAATTC.
2. A staggered cut leaves sticky ends: short unpaired bases that pair with complementary sticky ends.

• Reverse transcriptase makes cDNA from mRNA, for example insulin mRNA from pancreatic β cells.
• cDNA has no introns, which bacteria cannot remove.
• A gene can also be synthesised because its base sequence is known.

1. Cut the plasmid and the gene with the same restriction enzyme, so the sticky ends are complementary.
2. DNA ligase joins the sugar-phosphate backbones.
3. Calcium ions and heat shock help bacteria take up the plasmid; they are now transformed.

1. The gene is inserted into the ampicillin resistance gene, which stops it working.
2. On tetracycline agar, only bacteria with a plasmid grow.
3. Colonies that are missing on ampicillin agar carry the recombinant plasmid; pick them from the first plate.

Human insulin from bacteria

1. 1) Obtain the gene
2. 2) Insert into plasmids
3. 3) Transform E. coli
4. 4) Select with markers
5. 5) Grow in fermenters
6. 6) Purify the insulin

• For: human insulin in large amounts, fewer immune reactions and no animals needed.
• Against: antibiotic resistance marker genes could spread to pathogens, and modified bacteria could escape.

1. Remove the tumour genes from the Ti plasmid and insert the chosen gene.
2. The bacteria infect plant cells and the gene joins a plant chromosome.
3. Transformed cells grow into whole plants in tissue culture.

• Bt crops such as tomatoes and maize make an insecticidal protein from Bacillus thuringiensis.
• Herbicide-resistant soya survives glyphosate, so weeds can be sprayed.

• For: higher yields, less insecticide, more nutritious food, and crops that make medicines.
• Against: pollen may spread genes to wild relatives or organic crops, pests may evolve resistance, beneficial insects may be harmed, and the effects of new proteins are uncertain.

• Gene therapy inserts a functional allele into cells, or corrects the faulty one.
• Somatic therapy changes body cells and is not inherited; germ line therapy would be inherited.
• Vectors: disabled viruses, liposomes, plasmids, or gene editing such as CRISPR-Cas9.
• Cystic fibrosis: liposomes with the CFTR allele are inhaled, but treatment must be repeated.
• Sickle cell disease: the patient's own bone marrow stem cells are corrected and returned.

1. A deletion in the dystrophin gene shifts the reading frame, so no dystrophin is made.
2. An antisense oligonucleotide (molecular patch) binds to a chosen exon in the pre-mRNA, which is then skipped.
3. The reading frame is restored and a shorter, partly functional dystrophin is made.
4. Treatment must be repeated because the DNA is not changed.

• Faster, more accurate diagnosis of rare diseases.
• Choosing drugs and doses to suit a patient's genes, for example warfarin.
• Targeted cancer drugs, for example for tumours that over-express HER2.
• Screening people at high genetic risk.

1. Totipotent (zygote): any cell, including the placenta.
2. Pluripotent (embryonic stem cells): any body cell.
3. Multipotent (adult stem cells, for example in bone marrow): a few related cell types.
4. Induced pluripotent stem cells: adult cells reprogrammed to be pluripotent.

1. Cells, ideally the patient's own, are multiplied in culture.
2. They are seeded on a porous, biodegradable scaffold and the tissue is implanted.

• Against embryonic stem cells: the embryo is destroyed, and fears of reproductive cloning.
• For: embryos are under 14 days old, often spare IVF embryos, and the benefits could be great.
• The patient's own adult or iPS cells mean less rejection and fewer ethical concerns.

• 95 °C breaks hydrogen bonds; Taq withstands 95 °C.
• DNA moves to the positive electrode; small fragments go furthest.
• Same restriction enzyme for gene and plasmid; ligase joins backbones.
• Somatic gene therapy is not inherited.
• Exon skipping makes a shorter, partly working dystrophin.