Flashcards in Genome Manipulation Deck (67)
Define the term “DNA sequencing”.
Working out sequences of bases in a strand of DNA.
Define the term “terminator bases”.
Modified version of four nucleotide bases which stop DNA synthesis when they are included.
Define the term “high-throughput sequencing”.
New methods of DNA sequencing that are automated, very rapid and much cheaper than original techniques.
Describe the steps in DNA sequencing (the capillary method).
1) DNA mixed with a primer, DNA polymerase, an excess of normal nucleotides and terminator bases.
2) The mixture is placed in a thermal cycler that rapidly changes temp at programmed intervals. At 96oC the double stranded DNA separates into single strands (H-bonds between bases broken) and at 50oC the primers anneal (attach) to the DNA strand.
3) At 60oC DNA polymerase starts to build up new DNA strands by adding nucleotides with complementary bases to the single-strands of DNA.
4) Each time a terminator base is added instead of a normal nucleotide, the synthesis of DNA is terminated as no more bases can be added. Chain terminators bind at random so this results in many different size fragments of DNA. After many cycles, all of the possible DNA chains will be produced with reaction stopped at every base.
5) The DNA fragments are then separated according to their length by capillary sequencing. The fluorescent markers on each base are used to identify the final base on each fragment. Lasers detect the different colour and thus the order of the sequence.
6) The order of bases in the capillary tube shows the base sequence of the DNA strand which is complementary to the original strand of DNA.
7) Data from the sequencing process is fed into a computer that the reassembles the genomes by comparing all the fragments and finding the overlaps. This allows us to assemble the entire genome.
What is the purpose of genome sequencing?
Medical researches want to identify regions of the genome that are linked with particular diseases.
Can help us to discover evolutionary links.
Describe the reasons for developing new DNA sequencing technologies.
- More efficient, very fast.
- 3 billion base pairs of the human genome can be sequenced in just days.
Define the term “bioinformatics”.
The development of software and computing tools needed to analyse and organise raw biological data.
Define the term “computational biology”.
The study of biology using computational techniques to analyse large amounts of data.
Define the term "genome".
All the genetic material of an organism.
Define the term “DNA barcoding”.
The identification of particular sections of the genome that are common to all species but vary between them, so comparisons can be made.
Define the term “synthetic biology”.
The design and construction of novel biological pathways or devices or the re-design of existing natural biological systems.
Define the term "proteomics"
The study of amino acid sequencing of an entire an organism's protein complement.
Define the term “epidemiology”.
The study of how often diseases occur in different groups of people and why.
Explain why new DNA sequencing methods are allowing genome-wide comparisons between individuals and between species.
Genome analysis provides scientists with another tool to aide in species identification, by comparison to a standard sequences for a species.
Explain why the comparison of many human genomes may help the understating and treatment of human illness.
- Reveals patterns in the DNA and the diseases to which we are vulnerable.
- Enormous implications for the field of medicine.
- Identify changes in gene which cause a genetic disease and our likelihood of developing certain disease.
Describe 4 reasons why scientists may want to analyse the genomes of pathogens.
- Find the source of infection.
- Identify anti-biotic resistant strains of bacteria, ensuring antibiotics are only used when they will be effective and helping prevent the spread of antibiotic resistance.
- Scientists can track the outbreak of a potentially serious disease.
- Identify regions in the genome of pathogens that may be useful targets in the development of new drugs.
Describe how DNA sequencing allows scientists to identify the evolutionary relationships between species.
- DNA sequences of different organisms can be compared.
- Basic mutation rate of DNA can be calculated so scientists can calculate how long ago two species diverged from a common ancestor.
- Build up evolutionary trees.
Explain why, in theory, knowing a DNA sequence should allow you to identify the sequence of amino acids in the protein that the DNA sequence codes for. Also, explain why, in practice, this doesn’t always provide the correct sequence of amino acids in the protein.
- Because we traditionally thought that genes code for a particular protein, but we now know that the number of coding genes in the human DNA is very different the number of unique proteins.
- The expressed sequence of amino acids is not always what would be predicted from the genome sequence alone.
- Some genes can code for many different proteins.
Describe 4 techniques that could be classified as “synthetic biology”. Describe the role of DNA sequencing in each technique.
1) Genetic engineering - may involve a single change in a biological pathway or relatively major genetic modification of an entire organism.
2) Use of biological systems or parts of biological systems in industrial contexts, for example, the use of fixed or immobilised enzymes and the production of drugs from microorganisms.
3) The synthesis of new genes to replace faulty genes, r.g the faulty genes that cause cystic fibrosis.
4) The synthesis of an entire new organism, i.e. creating an artificial genome.
Define the term “DNA profiling”.
Producing an image of the patterns in the non-coding DNA of an individual.
Define the term “exon”.
Regions of coding DNA or RNA.
Define the term “intron”.
Regions of non-coding DNA or RNA that are removed from mRNA before it is translated.
Define the term “locus”.
The location of a gene on a chromosome.
Define the term “variable number tandem repeat” (VNTR).
- Short sequences of DNA repeated many times which vary greatly in number between individuals.
Define the term “minisatellite".
A region where a sequence of 20-50 base pairs (satellite DNA) will be repeated from 50 to several hundred times.
These occur at more than 1000 locations in the human genome.
Define the term “short tandem repeat”.
- A microsatellite is a smaller region of just 2-4 base pairs repeated only 5-15 times.
- Known as short tandem repeats.
Define the term “microsatellite”.
- A smaller region of DNA just 2-4 base pairs long and repeated only 5-15 times.
Describe how STRs vary and why they can be used to identify individuals.
- These satellites always appear in the same positions on the chromosomes, but the number of repeats of each mini/micro satellites varies between individuals, as different lengths of repeats are inherited from both parents.
- Only identical twins will have an identical satellite pattern.
- The more closely related you are to someone, the more likely you are to have a similar satellite pattern.
Name the 5 main stages in DNA profiling.
1) Extraction DNA.
- DNA is extracted from the sample.
2) Digesting the sample.
- Restriction endonucleases cut the DNA into fragments.
3) Separating the DNA fragments.
- DNA fragments are transferred from the gel to the nylon membrane in a process known as southern blotting.
- DNA probes are added to label the fragments.
- These radioactive probes attach to specific fragments.
5) Seeing the evidence.
- Membrane with radioactively labelled DNA fragments is place onto an x-ray film.
- Development of the x-ray film reveals dark bands where the radioactive or fluorescent DNA probes have attached.