M6C21 - Manipulating genomes Flashcards
What is a genome?
All of the alleles possessed by an organism.
What is an intron?
Non-coding regions of a gene that don’t contain codons to make a final protein.
What is an exon?
Regions of the gene that code, they contain codons that are later read to make proteins.
What is a restriction enzyme (restriction endonuclease)?
Enzymes that cut a DNA molecule at a particular sequence of bases.
What is satellite DNA (Introns)?
Short sequences of DNA that are repeated many times, the number of times that this repeats produces the variations in individuals.
What is a minisatellite region?
A sequence of 20-50 base pairs that are repeated 50-100 times.
Also known as variable number tandem repeats (VNTRs).
What is a microsatellite region?
A smaller region of just 2-4 base pairs repeated only 5-15 times. Also known as short tandem repeats (STRs).
Describe the extraction process in making a DNA profile:
DNA is extracted from the sample (blood, hair etc), polymerase chain reaction is used to give enough DNA to develop a profile.
What is a restriction site?
A specific nucleotide sequence where restriction endonucleases cut.
What is a VNTR (Variable number tandem repeat)?
Regions found in the non-coding part of DNA. They contain variable numbers of repeated DNA sequences.
Describe the digestion process in making a DNA profile:
-Strands of DNA are cut into small fragments using restriction endonucleases.
-Different endonucleases cut DNA at a specific restriction site.
-A mixture of restriction enzymes is used that leaves repeating units or satellites intact, so the fragments at the end of the process include a mixture of intact satellite regions.
Describe the separation process in making a DNA profile:
-Cut DNA fragments are separated using GEL ELECTROPHORESIS.
-Gel is then immersed in alkali to separate the DNA double strands into single strands.
Describe the hybridisation process in making a DNA profile:
-Radioactive or fluorescent DNA probes are added.
-DNA probes are short DNA or RNA sequences complementary to a known DNA sequence.
-Probes bind to complementary strands.
Describe the transfer (visualisation) process in making a DNA profile:
-Single-stranded fragments are transferred from the gel to nylon membrane (Southern Blotting).
-A thin nylon membrane is laid over the gel.
-Membrane is covered with sheet of absorbent paper to draw up the liquid through capillary action.
-This transfers the DNA to the nylon in the same positions they were on the gel.
Describe the development process in making a DNA profile:
-Excess probe is washed off.
-Nylon membrane with radioactively labelled fragments is placed onto x-ray film.
-Regions where probes have bound will develop the x-ray.
-Probes identify the microsatellite regions (more varied than minisatellite regions).
-The fragments give a pattern of bars (DNA profile).
Give uses of DNA profiling:
-Solving crimes
-Paternity testing
-Evolutionary relationships (how closely related organisms are).
-Genetic screening (genetic diseases).
Describe the use of DNA profiling in paternity testing
By comparing the DNA profile of a mother and her child we can identify DNA fragments in the child which are absent from the mother so must have been inherited from the biological father.
What is DNA sequencing?
Working out the sequence of nucleotides
What are terminator bases?
Modified versions of the four nucleotides which stop DNA synthesis when they are included.
Describe the principles of DNA sequencing developed by Frederick Sanger:
1) The DNA for sequencing is mixed with a primer, DNA polymerase, an excess of normal nucleotides, and terminator bases.
2) The test tubes are incubated at a temperature that allows the DNA polymerase to function.
The primer anneals to the start of the single stranded template (96 degrees), producing a short section of double stranded DNA at the start of the sequence.
DNA polymerase attaches to this double stranded section and begins DNA replication using the free nucleotides in the test tube.
3) At any time, DNA polymerase can insert one of the terminator bases by chance which results in the termination of DNA replication. Because the point at which the terminator base is inserted varies with every strand, complementary DNA chains of varying lengths are produced.
4) Once the incubation period has ended the new, complementary, DNA chains are separated from the template DNA.
The resulting single-stranded DNA chains are then separated according to length using gel electrophoresis
Describe automated sequencing:
Automated DNA sequencing uses computers.
-Nucleotides are labelled with fluorescent dyes.
-Everything occurs in only a single tube.
-DNA fragments are separated by their length by capillary sequencing, which works like gel electrophoresis.
-The fluorescent markers on the terminator bases are used to identify the final base on each fragment.
-Lasers detect the different colours and order the sequence.
-Computer analysis gives the original DNA sequence.
Describe the process of polymerase chain reaction:
1) Separating the strands:
The temperature is increased to 90 degrees, this denatures the DNA by breaking the hydrogen bonds so the strands separate.
2) Annealing the primers:
The temperature is decreased to 55 degrees and the primers bind to the ends of the DNA strands. They are needed for the replication of the strands to occur.
3) Synthesis of DNA:
The temp is increased to 72 degrees, which is the optimum temp for DNA polymerase to work best. DNA polymerase adds bases to the primer, building up complementary strands of DNA and so producing double-stranded DNA identical to the original sequence.
The enzyme TAQ POLYMERASE is used, which is obtained from thermophilic bacteria.
Explain the role of PCR in DNA analysis:
PCR allows rapid amplification of a specific segment of DNA.
It can be used in solving crimes as usually only small amounts of DNA are available at crime scenes. So PCR allows scientists to produce a lot of DNA from a small sample.
Describe why genome sequencing is used:
The chain termination is used for fragments up to 750 base pairs long. In order to sequence a whole genome you need to chop up the DNA first, then sequence fragments individually.
Describe high-throughput sequencing:
-FASTER than previous sequencing techniques, cheaper.
-One example is pyrosequencing. It doesn’t use electrophoresis.
What is gel electrophoresis and how does it work?
In electrophoresis, the DNA molecules are separated according to their size.
-DNA is NEGATIVELY charged due to the phosphate groups so are attracted to the anode.
-Fragments move through the gel based on their size, the smaller fragments move the fastest.
How are the DNA samples prepared for electrophoresis?
-DNA samples are collected and then amplified using PCR.
-Restriction endonucleases are then used to cut the DNA into fragments.
-Samples are prepared with DNA binding dyes.
Explain the practical process of gel electrophoresis:
-The DNA solutions are placed into wells in a layer of agarose gel. The gel is also covered with a buffer solution to maintain a constant pH.
-Electrodes are placed in either side of the gel, with the cathode placed at the end with the DNA samples.
-When an electric current is passed through the gel, the DNA fragments move through the gel towards the anode.
What is genetic engineering?
Genetic engineering involves altering an organism’s genome.
What is recombinant DNA?
Formed by combining DNA from two or more sources.
What is a transgenic organism?
An organism that contains nucleotide sequences from a different species.
What is a genetically modified organism?
Any organism that has introduced genetic material.
Describe the process of isolating genes for genetic engineering:
-Restriction endonucleases are required to cut the required gene from DNA.
What are ‘sticky ends’?
Endonucleases used to isolate genes often cut the two DNA strands unevenly, leaving one strand of the fragment a few bases longer.
-These regions with unpaired, exposed bases are called sticky ends and make it easier to insert the desired gene into a different organism.
What is a vector?
A DNA molecule that is used to transport a particular DNA segment into a host cell.
What are most commonly used as vectors in genetic engineering?
Plasmids or viruses
What is a plasmid?
Small, circular, double-stranded DNA molecules, distinct from the chromosomal DNA.
Explain how DNA fragments are inserted into plasmids to create recombinant DNA: ***
1)The plasmid is cut open using the same restriction endonuclease as used to isolate the DNA fragment, this means the plasmid will have complementary sticky ends to the sticky ends of the DNA fragment.
-Once the complementary bases of the two sticky ends are lined up, DNA LIGASE forms phosphodiester bonds between the sugar and the phosphate group on the two strands.
What is reverse transcriptase and what does it do?
Reverse transcriptase is an enzyme that can turn RNA (specifically mRNA) into DNA so that it can be transcribed by the host cell into proteins.
-USED to obtain a desired gene fragment.
-Used by genetic engineers as mRNA is easier to extract than and the complementary DNA (cDNA) formed doesn’t contain introns (non-coding regions).
Describe the method in which plasmids are transferred into host cells:
Electroporation is where a small electrical current is applied to the bacteria which makes the membranes porous and so the plasmids move into the cells. It can also be used to get DNA fragments directly into eukaryotic cells.
What is a marker gene and why is it used?
Marker genes are added to a vector along with the gene of interest that produce a noticeable change in the cell e.g. antibiotic resistance.
-USED to indicate whether the bacteria has taken up the plasmid.
Explain how scientists can use the marker gene of antibiotic resistance to determine the success of transferring a vector:
Grow the bacteria on agar containing antibiotic.
Discuss the ethical issues with insect resistance in genetically modified soya:
- = Encourages monoculture which reduces biodiversity.
+ = Increases the crop yield as plants are less likely to be destroyed by insects.
What is ‘pharming’ and what are the ethical issues with it:
Pharming is where scientists genetically modify livestock to produce pharmaceutical drugs. E.g. biopharm sheep and goats are GM to produce a number of useful human proteins in their milk.
-Animals are unable to consent.
-Mutations could occur which would be harmful for the animals.
Explain GM in pathogens and ethical issues surrounding it:
Animal and plant pathogens can be modified to observe their drug resistance and how much damage they cause to its host.
-Transferred genes could spread to wild populations and cause problems e.g. superweeds.
-GM of bacteria could increase antibiotic resistance.
Explain how genetic modification can have negative effects for farmers:
Biotech companies charge farmers more money for GM seeds to make back the money invested in their product.
Seeds can’t be regrown every year.
Purchasing new seeds each year presents financial issues for farmers.
Describe the process of Somatic gene therapy:
1) Cells are harvested from the patient.
2) Viruses are altered so they can’t reproduce.
3) A gene is inserted into a virus.
4) Altered virus mixed with patient’s cells.
5) Cells become transgenic.
6) Altered genes are injected into the patient’s body.
7) Altered cells produce the desired protein.
Describe the process of Germline gene therapy:
1) Functional gene is extracted from plasmid/vector.
2) Functional gene is inserted into early embryo.
3) Embryo cells containing the functional gene grow by mitosis.
4) Healthy baby has all cells with functional gene.
Compare the differences between somatic and Germline therapy
-In Somatic gene therapy changes aren’t heritable and are confined to the individual whereas in Germline gene therapy changes are heritable and will be passed on.
-In Somatic, gene is introduced into a non-reproductive cell whereas in Germline gene is introduced into a gamete.
-Somatic therapy is short term and needs repeating whereas Germline is long-term and permanent.
Why is the chain termination technique best to use when sequencing a human gene that causes a genetic disease?
Only 5 errors in 100,000 nucleotides compared to 50 in pyrosequencing.
What is bioinformatics?
The development of software and computing tools needed to organise and analyse raw biological data. E.g. development of algorithms, statistical tests.
What is computational biology?
The study of biology using computational techniques, especially in the analysis of huge amounts of biodata.
Uses data from bioinformatics to build theoretical models of biological systems, which can be used to make predictions.
How can DNA sequencing impact epidemiology (medicine)?
Computers can analyse and compare genomes of individuals, revealing patterns in the DNA we inherit and diseases to which we are vulnerable.
What does using genome-wide comparisons to analyse the genomes of pathogens enable?
-Doctors to identify antibiotic-resistant strains of bacteria.
-Doctors to find the source of an infection e.g. bird flu, MRSA.
Describe how genome-wide comparisons are used to identify species:
DNA Barcoding = Identifying sections of the genome common to all species but vary between them for comparisons.
Scientists identify using short sections of DNA from a conserved region of the genome.
Describe how genome-wide comparisons are used to search for evolutionary relationships:
DNA sequences of different organisms can be compared, because basic mutation rate of DNA can be calculated and scientists can calculate how long ago 2 species diverged from a common ancestor.
Evolutionary trees can be built.
What is proteomics?
The study and amino acid sequencing of an organism’s entire protein complement.
Gives us knowledge into the relationship between genotype and phenotype.
Describe the function of splicesomes in proteomics:
-mRNA transcribed from DNA in the nucleus includes introns and exons.
-Before translation pre mRNA is modified: Introns are removed, exons are joined by SPLICESOMES to give mature functional mRNA.
Describe protein modification in proteomics:
-Proteins are modified after synthesis.
-A protein coded by a gee may remain intact or be shortened or lengthened to give a variety of other proteins.
What is synthetic biology?
Design and construction of novel artificial biological pathways, organisms or devices, or the re-design of existing biological systems.
Give some techniques within synthetic biology:
-Genetic engineering
-Synthesis of new genes to replace faulty ones.
