Manipulating genomes Flashcards
what is a genome of an organism?
all the genetic material it contains- in eukaryotes, it is the DNA in the nucleus and mitochondria combined
What are exons?
Regions of DNA which code for proteins
What are introns?
Non-coding regions of DNA that are removed from mRNA before translation occurs. They are involved in gene expression control.
what is satellite DNA?
short repeated DNA sequences found in introns and centromeres.
what are the different types of satellite DNA?
minisatellite DNA (VNTRs) and microsatellite DNA (STRs)
describe minisatellite DNA (VNTRs)
-sequence of 20-50 base pairs
-repeated 50-100 times
describe microsatellite DNA (STRs)
-sequence of 2-4 base pairs
-repeated 5-15 times
What is the principle for DNA profiling?
different people have different numbers of repeats and therefore will generate different satellite patterns
What is DNA profiling?
producing an image of the patterns in the DNA. Technique helps to identify individuals and familial relationships.
How do you produce a DNA profile?
1) Extract the DNA and use polymerase chain reaction (PCR) technique to amplify the DNA sample to develop a profile.
2) Digest the sample into small fragments by using restriction endonuclease enzymes. They use a mixture of restriction enzymes that leave satellites intact, so the fragments at the end of the process include a mixture of intact mini- and micro-satellite regions
3)DNA fragments are separated using electrophoresis and southern blotting occurs
4) Hybridisation occurs using radioactive or fluorescent DNA probes
Describe how restriction endonuclease enzyme works
different restriction endonucleases cut DNA at a specific nucleotide sequence, known as restriction site or recognition site. All restriction endonucleases make two cuts, once through each strand of the DNA double helix.
what is electrophoresis?
a technique that utilises the way charged particles move through a gel medium under the influence of an electric current
Why is the electrophoresis gel immersed in alkali?
in order to separate the DNA double strands into single strands
What is southern blotting?
when single DNA strands are transferred onto a membrane
Describe the process of hybridisation when producing a DNA profile
-DNA probes are added to the excess DNA fragments on the membrane
-DNA probes are short DNA or RNA. sequences complementary to a known DNA sequence. This requires specific pH and temp
-DNA probes identify the micro-satellite regions that are more varied than the larger mini-satellite region. The excess probes are washed off.
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How can you view the evidence of a DNA profile?
-if a radioactive DNA probe was used, X-ray images are taken of the membrane
-if a fluorescent DNA probe was used, UV light would be used
what are the main steps in the polymerase chain reaction?
-denaturation (95 degrees), double helix separated
-annealing (55 degrees), primers bind to the start of the target DNA
-synthesis (72 degrees), building up the new strand
what is a thermo cycler?
a machine that carefully controls and changes its temperature at programmed timings to trigger different steps in the PCR. It is also used in DNA sequencing.
Describe the denaturation step of the PCR?
At 95 degrees, molecules gain kinetic energy to which they vibrate more and break the hydrogen bonds between the complementary bases.
what are primers?
short DNA sequences which bind to the start of the gene that you want to amplify
Describe the annealing step of the PCR?
At 55 degrees, primers bind (anneal) to the ends of thee DNA strand- complementary base pairing
Describe the synthesis step of the PCR?
At 72 degrees, free nucleotides pair up to exposed bases by complementary base pairing. Taq polymerase joins up sugar-phosphate backbone by forming phophodiester bonds
Why is Taq polymerase used instead of human DNA polymerase?
human DNA polymerase would denature however Taq polymerase can withstand high temperatures as it is obtained from thermophilic bacteria found in hot springs
what are the uses of DNA profiling?
-used in forensic science to identify criminals
-used to determine the paternity of a child
-used to identify children who are at risk of developing a certain disease
What are the steps in the DNA sequencing process?
1) DNA sample sequenced
2)free nucleotides
3)coloured florescent-labelled terminator bases
4)DNA polymerase
5)DNA primers
Explain the first step of DNA sequencing?
-heat up to 95 degrees for denaturation
-cool down to 55 degrees for the primers to anneal
-heat to 60 degrees in which DNA polymerase acts to form phosphodiester bonds
What is a terminator base?
It terminates DNA synthesis and no more bases can be added. Terminator bases have a hydrogen atom instead of a hydroxyl group at the 3’ carbon of the ribose ring. They cannot form phosphodiester bonds with the next nucleotide.
How are all of the possible DNA chains produced during gene sequencing?
as the chain-terminating bases are present in lower amounts and added at random, this results in many DNA fragments of different lengths depending on where the chain terminating bases have been added during the process. Cycle is repeated many times.
What are the coloured fluorescent tags for terminator bases?
A- green
G-yellow
T-red
C-blue
How are the DNA fragments separated in DNA sequencing?
DNA fragments are separated according to their length by capillary sequencing, which works like gel electrophoresis in minute capillary tubes. Lasers detect the different colours (fluorescent tag) and thus the order of the sequence.
Describe the principles of electrophoresis after DNA sequencing?
-pipette the four solutions containing the four different terminator bases into the different wells
-pass a current through the electrophoresis plate from - to +
-DNA has a slightly negative charge so it will be repelled by the cathode (-) and attracted to the anode (+)
-smaller fragments will travel further as they have less mass and therefore less resistance in the gel
What are the two ways to see DNA after electrophoresis?
1) Southern blotting using radioactive DNA probes and x-rays
2)Using a GFP (green fluorescent protein) DNA probe and UV light
Once the genome is assembled, what is it used for?
-scientists want to identify the gene that codes for a specific characteristic
-medical researchers want to identify regions which are linked to specific diseases
What is the faster technique for DNA sequencing called?
Massive parallel sequencing- clusters of DNA fragments are sequenced and imaged at the same time
Describe how gene sequencing now occurs?
High throughput sequencing and next generation sequencing where multiple sets of DNA is sequenced together (massively parallel sequencing)
Describe how gene sequencing now occurs?
High throughput sequencing and next generation sequencing where multiple sets of DNA is sequenced together (massively parallel sequencing)
What is bioinformatics?
This is where software is developed to process and understand large complex data (DNA sequences) using computational biology. It allows access to large amounts of data.
What is computational biology?
uses data from bioinformatics to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances.
Describe bioinformatics using computational biology?
-access to large amount of data on DNA and proteins
-information is universal
-allows rapid comparison of sequences with newly sequenced alleles
-amino acid sequence/ protein structures held in database
-computer modelling of new protein structure from base sequence
What is genomics?
The field of genetics that applies DNA sequencing methods and computational biology to analyse the structure and function of genomes
what is epidemiology?
Epidemiology is the method used to find the causes of health outcomes and diseases in populations
what is epidemiology?
Epidemiology is the method used to find the causes of health outcomes and diseases in populations
Why is genomics significant in epidemiology?
It reveals patterns in the DNA we inherit and the diseases to which we are vulnerable.
Sequencing of genomes enables (it is fast and cheap)…
-Doctors can find out the source of an infection
-Doctors can identify antibiotic-resistant strains of bacteria, ensuring antibiotics are only used when they will be effective and preventing the spread of antibiotic resistance.
-scientists can track and monitor the progress of an outbreak
-scientists can identify regions in the genome of pathogens that may be useful targets in the development of new drugs and to identify genetic markers for use in vaccines
What is DNA barcoding?
a useful technique to identify particular sections of the genome that are common to all species but vary between them, so comparisons can be made
How does DNA sequencing enable scientists to build up evolutionary trees?
As the DNA sequence of different organisms can be compared, the basic mutation rate of DNA can be calculated scientists can calculate how long ago two species diverged from a common ancestor.
what is proteomics?
the study and amino acid sequencing of an organisms entire protein complement.
Describe proteomics?
some genes can code for many different proteins therefore the sequence of amino acids is not always what would be predicted. There is a complex relationship between genotype and phenotype.
How can one gene code for different proteins?
-the introns are removed from pre-mRNA (mRNA before translation)
-exons to be translated are joined together by spliceosomes to give functional mRNA.
-spliceosomes can join exons in a variety of different ways so therefore a single gene may produce several versions of functional mRNA- coding different arrangements of amino acids and therefore different phenotypes
what is protein modification?
a protein that is coded by a gene may remain intact or may be shortened or lengthened to give a variety of other proteins
what is synthetic biology?
field of science that involves redesigning organisms for useful purposes by engineering them to have new abilities
what techniques does synthetic biology include?
-genetic engineering
-biotechnology
-synthesis of new genes to replace faulty ones
-synthesis of an entire new organism
why do we compare genomes?
-evolutionary relationships (phylogeny)
-compare base sequence to predict the function of an unknown protein
-universal
-compare between and within species
What is the first step in genetic engineering?
restriction endonucleases are used to cut the desired gene from DNA. This creates sticky ends which make it easier to insert desired genes.
What is another technique to isolate a desirable gene?
isolating the mRNA for the desired gene and use reverse transcriptase to produce a single strand of complementary DNA. The advantage of this technique is that it makes it easier to identify the desired gene.
what is genetic engineering?
manipulation of an organism’s genome to achieve a desired outcome
what is a transgenic organism?
organism that carries a gene from another organism
what is the second step in genetic engineering?
the plasmid (vector) is cut using the same restriction enzyme to produce complementary sticky ends
what is the most common vector used in genetic engineering?
bacterial plasmids- small circular molecules of DNA separate from the chromosomal DNA that can replicate independently.
What is the third step of genetic engineering?
The desired gene is inserted into the vector/ plasmid using enzyme DNA ligase. The desired gene will be inserted with a marker e.g fluorescent marker or a gene that codes for antibiotic resistance
What is the fourth step of genetic engineering?
The recombinant DNA (plasmid carrying desired gene) is inserted into the host cell using electroporation. This is where an electric shock makes the membrane porous and therefore the plasmids can pass through the membrane of the host cell
what is another way that the recombinant DNA is inserted into the host cell (transformation)?
culture the bacterial cells and plasmids in a calcium-rich solution and increase the temperature. This causes the bacterial membrane to become permeable and plasmids can enter.
What is the fifth step of genetic engineering?
The host cells undergoes mitosis, reproducing the desired gene too. This is then tested to ensure the desired gene has been taken up by the host cell by looking at fluorescence or applying antibiotic (dependent on marker used)
How is electrofusion used to produce GM organisms?
Tiny currents are applied to the membrane of two different cells. This fuses the cell and nuclear membrane of two different cells together to form a hybrid or polyploid cell. This is most effective in plant cells compared to animal cells as polyploid mammalian cells usually don’t survive.
what vector can be used for plants?
Agrobacterium tumefaciens (bacterium that causes tutors in healthy plants), plasmids, virus
What vector can be used for bacteria?
BAC(bacteria artificial chromosome), bacteriophage, virus
What vector can be used for animals?
Virus, BAC, plasmid
For what reason are prokaryotes genetically engineered?
to produces substances that are useful to people e.g insulin, human growth hormone, antibiotics, pure vaccines and many of the enzymes used in industry
For what reasons are plants genetically engineered?
for pesticide production, herbicide resistance, drought-resistance or higher yield
What plasmid is the desired gene placed in A. tumefaciens?
Ti plasmid along with a marker gene
Ethical considerations include…
human rights, human health and safety, animal welfare, and the protection of the environment
What is the disadvantage of using antibiotic resistance as a marker gene?
risk that this resistance could spread to wild populations
What are the advantages of GM crops?
-pest resistance reduces amount of pesticide spraying and protects environment, increasing yield
-disease resistance reduces crop losses, increasing yield
-herbicides can be used to reduce competing weeds and increase yield
-the extended self life of some GM crops reduces food waste
-crops can grow in a wider range of conditions/ survive adverse conditions
-nutritional value of crops can be increased
-plants could be used to produce human medicines and vaccines
Disadvantages of GM crops?
- non-pest insects and insect-eating predators might be damaged by the toxins in GM plants
-insects pests may become resistant to pesticides in GM crops
-transferred genes might spread to wild populations and cause problems e.g superweeds
-biodiversity could be reduced if herbicides are overused to destroy weeds
-extended self life may reduce the commercial value and demand for the crop
-people may be allergic to the different proteins made in GM crops
what is pharming?
genetic engineering in animals for the production of human medicines
what are the two aspects of the field of pharming?
-creating animal models: the addition or removal of genes so that animals can develop certain diseases, acting as models for the development of new therapies
-creating human proteins: the introduction of a human gene coding for a medically required protein. Animals are sometimes used because bacteria cannot produce all of the complex proteins made by eukaryotic cells
What are the two types of gene therapy?
-somatic cell gene therapy
-germ line cell gene therapy
Describe somatic cell gene therapy?
This involves replacing the mutant allele (mutant gene causing disease) with a healthy allele in the affected somatic cells
Describe germ line gene therapy?
-inserting a healthy allele into the germ cells- usually the eggs- or into an embryo straight after fertilisation
-the individual would be born healthy with normal allele in place- and would pass it on to their offspring
what is gene therapy?
Gene therapy is the repair or replacement of faulty genes with healthy versions
What are the problems with germ line therapy?
-without consent of the unborn individual
