Manipulating Genomes Flashcards
What is the genome of an organism?
All the genetic material it contains.
What is a proteome? What is needed to know it?
All the proteins a cell can produce. For simpler organisms, knowing the genome sequence means the proteome can be derived from the genetic code.
What are the applications of deriving the proteome from an organisms genome?
Identification of antigens for use in vaccine prodiction.
Why can knowledge of the human genome (and more complex organisms) not be easily translated into the proteome?
Because of the presence of non-coding DNA and regulatory genes.
How have DNA sequencing methods changed?
They are continually updated and have become automated. This has increased the speed of sequencing and allowed the whole genome to be sequenced through methods like high-throughput sequencing.
What is the basic principle of DNA sequencing?
- DNA polymerase, a primer, excess nucleotides, terminator bases and the DNA to be sequences are all mixed together.
- Mixture is in a thermocycler (used in PCR) to synthesise DNA
- DNA polymerase adds bases complimentary to DNA creating new DNA strand
- Terminator bases added at random, terminating sequence of DNA at diff point in each replicating strand.
- Continues until all the possible DNA chains will be produced with the terminator bases added at every possible position.
- Each terminator base (A T C G) are labelled with a different fluorescent colour so the end base of each DNA fragment can be identified.
- The DNA fragments are separated via gel electrophoresis according to length.
- As each fragment is arranged by length and the terminator base is fluorescent, the sequence of DNA can be worked out.
What is high-throughput sequencing?
When many DNA fragments are processed and sequenced simultaneously. This makes the process more efficient.
What has analysing pathogen’s genome resulted in?
- Identifying source of an infection
- Identifying antibiotic resistant bacteria (eg MRSA in hospitals)
- Tracking spread of pathogens to monitor potential epidemics and pandemics
- Identifying regions in the genome for new drugs to target.
What has comparing genomes improved?
- Accuracy of classification of sequences.
- Our understanding of evolutionary relationships.
What is Bioinformatics?
A field of biology that involves the storage, retrieval, and analysis of data from biological studies. Once a genome is sequenced, bioinformatics allows scientists to make comparisons with the genomes of other organisms
What is epidemiology?
The study of infectious disease within populations. The genomes of pathogens can be sequenced and analysed to aid research and disease control.
What is synthetic biology?
The creation of artificial pathways, organisms, devices or the redesign of natural systems. Sequencing genome has enabled this development.
Give examples of synthetic biology developments.
- Genetic engineering (Indulin-producing bacteria)
- Use of biological systems of industry (immobilised enzymes)
- Synthesis of new genes to replace faulty versions
- Synthesis of new organisms (eg new bacterial genomes)
What are VNTRs?
Variable number tandem repeats. These are what make up introns. Closely related = similar VNTR.
What is genetic fingerprinting?
The analysis of VNTR fragments to determine genetic relationships and genetic variability in a population.
Explain the process of DNA profiling.
- Collect sample of DNA from any cell with a nucleus. Eg hair follicle, body cells
- Use PCR machine to replicate lots of DNA so we can repeat this multiple times.
- Restriction endonucleases are added to cut DNA into smaller enzymes. They cut DNA at specific recognition sites close to target VNTRs.
- Separate cut up pieces of DNA via gel electrophoresis.
- DNA samples are loaded into small wells in agar gel. The gel is placed into buffer liquid with an electrical voltage applied.
6.DNA is negatively charged so moves to positive end of the gel. The shorter VNTRs move faster, separating the lengths. - An alkaline is added to separate the double strands of VNTR by breaking H bonds.
- DNA probes with a radioactive or fluorescent label form H bonds to complementary base sequence of VNTRs.
- Rinse gel and place under X rays to visualise position of radioactive gene probes, or under UV light to visualise fluorescent probe.
- Position of DNA bands are compared to identify genetic relationships, presence of a disease causing gene and to match unknown samples from a crime.
What is PCR?
Polymerase chain reaction. It is used to amplify the DNA by making millions of identical copies of a given DNA sample.
Wat is the equipment list of PCR?
Thermocycler, DNA fragment to be amplified, primers (short sequences of single strand of DNA needed to initiate DNA replication) , DNA polymerase (taq polymerase) and DNA nucleotides.
Why is taq polymerase used in PCR/
Because it doesn’t denature until very high temperatures. It is from bacteria that naturally grow in hotsprings.
Describe the method of PCR.
A reaction mixture is set up by mixing the DNA sample, primers, free nucleotides and DNA polymerase which is the enzyme involved in creating new DNA strands.
2) The mixture is then heated to 95 degrees to break the hydrogen bonds and to separate the two strands (denaturation)
3) The mixture is then cooled to between 50-60 degrees degrees so primers can bind to the strands (annealing)
4) Temperature is increased to about 72 degrees as this is the optimum temperature
DNA polymerase works at.
5) DNA polymerase creates a copy of the sample by complementary base pairing
using the free nucleotides (elongation)
6) This cycle is repeated around 30 times and gives rise to an amount of DNA sufficient to create a DNA profile.
What are the advantages of PCR?
Automated -efficient
Rapid - 100 billion copies of DNA can be made in hours
Doesn’t require living cells
What is genetic engineering?
The manipulation of the DNA sequences of an organism. Scientists have been able to artificially change an organism’s DNA by combining lengths of nucleotides from different sources
What is recombinant DNA?
Altered DNA with introduced nucleotides from different sources (nucleotides from different species).
What is a transgenic organism?
If an organism contains nucleotide sequences from a different species it is called a transgenic organism. It is genetically modified.
What are the uses of genetic engineering?
- increasing crop yield through resistance to drought, disease, pesticides and herbicides
-increased nutritional value (golden rice) - Give livestock disease and pest resistance
- Bacteria to produce medicines like insu;in or to decompose toxic pollutants or carry out large scale chemical production.
How are DNA fragments prepared to be inserted into vectors for genetic engineering?
- Restriction endonuclease cut at recognition sites of DNA, leaving sticky ends.
- Promotor region (RNA polymerase binding site) added at start of DNA fragment so transcription of these genes can occur.
- Terminator region added at end of fragment. So RNA polymerase will detach and only one gene at a time is transcribed to mRNA.
How is the DNA fragment inserted into a vector?
Plasmids are the most common vector. Circular DNA separate from bacterial genome.
Plasmid cut using restriction endonuclease, creating complimentary sticky ends to gene.
Mix the 2. Add DNA ligase to join plasmid and gene at sticky ends. Ligase catalyses condensation reaction to form phosphodiester bonds between gene and plasmid. This is now recombinant DNA.
How is the vector inserted into the host cell?
The host cell cell membrane needs to be more permeable. Host cells mixed with Ca2+ ions and they are heat shocked. So vector enters host cell’s cytoplasm. Not all bacteria get transformed.
How can you identify which cells are transformed in genetic engineering?
By the use of gene markers on the plasmids - fluorescent, enzymes or antibiotic resistance.
Why are not all host cells transformed during genetic engineering?
- Recombinant plasmid doesn’t get inside the cell
- Plasmid re-joins before the gene entered.
- DNA fragment sticks to itself rather than inserting into the plasmid.
Describe how antibiotic resistance markers used in genetic engineering work.
A plasmid from a bacteria resistant to ampicillin and tetracycline has the gene inserted into it. It disrupts one of those genes so it no longer makes a functional protein. Any bacteria that take up the plasmid will be resistant to one antibiotic and not the other. These are taken and cloned.
What are the perceived negatives of genetic engineering in plants?
The genes for pest, disease and herbicide resistance might spread to the wider environment and into other plants.
People may be allergic to the different proteins some crops may now make.
The technology is often patented so buying GMO seeds is unaffordable for poorer farmers.
How has genetic engineering been used in animals?
Modified viruses and gold-covered DNA has been injected into animals to carry new genes into their DNA.
As a result swine-flu resistant pigs and faster growing salmon have been created.
What is pharming?
The use of genetically modified animals to
produce pharmaceuticals
How has genetic engineering changed the soya crop industry?
Soya plants are a major crop world wide and at leat 50% are genetically engineered strains. One modification is the addition of a gene that allows Soya plants to produce Bt protein, which is toxic to pests.
What is gene therapy?
When human DNA is altered to treat disorders. There is potential to replace a faulty gene, inactivate a faulty gene or insert a new gene. Eg relace recessive Cystic fibrosis allele with dominant allele.
How would gene therapy work?
The cells would need to be isolated. Then a viral vector with desired allele inserted in its DNA would be used to insert the allele into the DNA from isolated human cells. The cels will be injected into the patient, producing desired protein.
What is the limitation of gene therapy?
The treatment is not a cure as it does not replace all the cells in the body with the faulty gene. It just supplements them. It is an example of somatic cell gene therapy.
What is somatic cell gene therapy?
Replacing body cells with cells with desired allele to treat disorders.
Ex vivo – the new gene is inserted via a virus vector into the cell outside the body. Blood or bone marrow cells are extracted and exposed to the virus which inserts the gene into these cells.
In vivo – the new gene is inserted via a vector into cells inside the body
What is germ line therapy?
The alteration of DNA in the gametes so the offspring would not inherit the faulty allele
