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.