Chapter 21 - Manipulating Genomes Flashcards
What is the genome?
All the genetic material in an organism
What are telomeres?
Sections of DNA found at the ends of each chromosomes
What are exons?
Coding DNA
What are introns?
Non-coding DNA
Where is satellite DNA found?
Introns
What are the two types of satellite DNA?
Minisatellite DNA
Microsatellite DNA
What is minisatellite DNA?
20-50 base pairs repeated 50-100 times
What is microsatellite DNA?
2-4 base pairs repeated 5-15 times
Why is satellite DNA useful?
It always appears in the same area on chromosomes, however different people have different numbers of repeats (different satellite patterns), so they can be used to identify an individual or determine familial relationships
What is the relationship between relation to an individual and differences in satellite DNA?
The more closely related you are to an individual, the more similar the satellite patterns
Give 2 uses of DNA profiling
- Match DNA at a crime scene to a suspect
- Determine familial relations e.g. find who the father of a child is
What is the first step of DNA profiling?
DNA extraction- a sample of DNA must be extracted. It may be a very small sample, however PCR can be used to amplify the DNA sample enough to develop a DNA sample suitable for profiling
What are the purpose of restriction endonucleases in DNA profiling?
Restriction endonucleases cut up the strands of DNA into smaller fragments. Different restriction endonucleases cut DNA at specific nucleotide sequences
What is the location in which the the restriction endonuclease cuts known as?
The restriction site
In DNA profiling, after the DNA is cut into small fragments, what must happen?
The fragments must be separated using gel electrophoresis
How does gel electrophoresis separate the DNA fragments?
DNA moves through the gel medium under the influence of an electric current. The DNA moves from the negative to the positive side, separating the strands fragments into a clear order/pattern
In electrophoresis, why does DNA move towards the positive end?
Because DNA contains a negative phosphate group
In gel electrophoresis, what is the gel immersed in and why?
The gel is immersed in alkali to separate the DNA double strands into single strands, to expose the bases of the DNA
What is southern blotting?
Where the fragments are transferred from the gel onto a nylon membrane
In DNA profiling, what is hybridisation?
Where radioactive or fluorescent DNA probes are added to the fragments and bind to complementary strands
What does the rate of movement through gel electrophoresis depend on?
The size of the DNA fragments. The smaller the fragment the more easily it can move through the gel
What is used to view the results in DNA profiling if radioactive probes were added?
If radioactive probes are used, X-ray images are taken
What is used to view the results in DNA profiling if fluorescent probes were added?
UV light will be shined on the results, making them glow
What is the result of the process of DNA profiling?
The fragments will give a pattern of bars, which is unique to every individual (except identical siblings)
What does PCR stand for?
Polymerase chain reaction
What is the first step of PCR?
Denaturation
What happens in the denaturation step of PCR?
The temperature is increased to 95C, which denatures the DNA by breaking the hydrogen bonds holding the strands together, so the strands separate
What is the step after denaturation in PCR?
Annealing of the primers
What is the temperature in the annealing of the primers step in PCR?
55C
What is the temperature in the denaturation step of PCR?
95C
What happens in the annealing of the primers step of PCR?
Primers bind (anneal) to the ends of the DNA strands
What is the step after the annealing of the primers?
Synthesis of DNA
In PCR, during synthesis of DNA why is the temperature raised to 75C?
Because this is the optimum temperature for DNA polymerase
What happens during the synthesis of DNA step of PCR?
DNA polymerase adds bases to the primers, building complementary strands of DNA and so producing double-stranded DNA identical to the original sequence
What type of DNA polymerase is used in PCR and why?
Taq polymerase is used, as normal human DNA polymerase would denature at the high temperature reached during PCR
What happens after the synthesis of DNA step of PCR?
The process repeats, this time with double the number of DNA strands as the cycle before. This results in an exponentially growing number of identical strands for each cycle
What 5 things do you need for DNA sequencing?
- DNA sample to be sequenced
- Free nucleotides in excess
- Coloured fluorescent labelled terminator bases
- DNA polymerase
- DNA primers
What is the first step of DNA sequencing?
It is heated to 95C, denaturing the DNA
What happens after the DNA denatures in DNA sequencing?
The mixture is cooled to 50C and primers are added
What is the role of DNA polymerase in DNA sequencing?
DNA polymerase is the enzyme that joins complementary nucleotides to the chain
What are terminator bases?
Bases that are missing an oxygen so they cannot form phosphodiester bonds with the next nucleotide. This means that no more bases can be added after a terminator bases
What is the role of terminator bases in DNA sequencing?
Each time a terminator base is added instead of a normal base, the synthesis of DNA for that chain is terminated
In DNA sequencing, what will eventually be produced produced after enough cycles?
Eventually, all possible DNA chain lengths will be produced, as the synthesis of the chain had been stopped at every base once
In DNA sequencing, what happens after all possible DNA chain lengths have been produced?
The fragments are separated by capillary sequencing and lasers detect the different terminator base colours
What machine is used to change the temperature in DNA sequencing and PCR?
Thermal cycler
What is the next generation sequencing technique for DNA sequencing?
The sequencing reaction takes place on a plastic slide known as a flow cell. This sequencing technique involves all of the clusters being sequenced and imaged simultaneously, so is much faster
2 uses of DNA sequencing of humans
Scientists can use sequence data to determine the function of different sections of DNA
Sequence data can highlight changes in a gene that may cause disease
What is bioinformatics?
The development of the software needed to organise and analyse raw biological data
What is computational biology?
The use of biological data in the building of theoretical models of biological systems, which can be used to predict what will happen in different circumstances
What are two reasons for the DNA sequencing of pathogen’s genomes?
- Doctors can identify antibiotic-resistant strains of bacteria, ensuring antibiotics will only be used when they will be effective
- Scientists can identify regions in the genomes of pathogens that may be useful targets in the development of new drugs
2 uses of DNA sequencing in organism classification
- DNA barcoding can be used to identify the species of an organism
- DNA sequencing can be used to understand evolutionary relationships between species
What is the main ethical concern with the genetic manipulation of microorganisms?
That the genetic engineering of pathogens could be used for purposes of biological warfare
What are 3 characteristics crops can be genetically engineered to have?
- Disease resistance
- Extended shelf-life
- Ability to survive adverse conditions
What is a pro of disease resistant crops?
Reduces crop losses from disease/increases yield
What is a con of disease resistant crops?
Engineered genes may spread to wild populations and cause problems e.g. superweeds
What is a pro of extended shelf life crops?
Reduces food waste
What is a con of extended shelf life crops?
May reduce the commercial value of the crop (could affect the livelihood of the farmers)
What is a worry of the use of genetically modified crops?
That people in less economically developed countries will be prevented from accessing and using them due to patents and issues with technology transfer
What is pharming?
The production of human medicines from the genetic engineering of animals and plants
What is gene therapy?
The introduction of normal genes into cells in place of missing or defective ones in order to correct genetic disorders
What is somatic gene therapy?
Where the mutant (faulty) allele is replaced by a healthy allele in the affected somatic (body) cells
What are 2 limitations of somatic gene therapy?
- Somatic cells have a limited life, and when they are replaced they will have the faulty allele again
- A treated individual will still pass the faulty alleles onto their offspring
What is germ line cell gene therapy?
The insertion of a healthy allele into the germ cells (normally egg cells), or into an embryo immediately after fertilisation. The child would be born with the healthy allele.
What is the advantage of germ line cell gene therapy?
The individual would have the correct healthy allele through their life, and pass the healthy allele onto their offspring
What are the concerns with germ line cell therapy?
- The potential impact on an individual of an intervention on the germ cells is unknown
- The technology may be a slippery slope, in which eventually people may choose desirable characteristics only for their offspring
What is the definition of transgenic?
Transgenic is a term that describes an organism containing genes from another organism put into its genome through recombinant DNA techniques
What are the two main methods of isolating the desired gene in genetic engineering?
Use of restriction endonucleases
Use of reverse transcriptase
How can you use restriction endonucleases to isolate a desired gene?
Restriction endonucleases cut at specific restriction sites, and can cut out the desired gene
What are sticky ends?
Restriction endonucleases cut the DNA strands unevenly, leaving unpaired exposed bases known as sticky ends
Why are sticky ends useful in genetic engineering?
Because as we use the same restriction endonucleases to cut both the desired gene and the plasmid, the complimentary sticky ends will bind to each other
How can you use reverse transcriptase to isolate a desired gene?
Reverse transcriptase will produce a complimentary single strand of DNA (cDNA) from the mRNA of the desired gene, giving you the correct base sequence
What is the most common vector in genetic engineering?
Plasmids
How is the desired gene inserted into the vector?
The complimentary sticky ends bind to each other, and DNA ligase forms phosphodiester bonds between the gene and plasmid
What is the product of the insertion of the gene into the plasmid known as?
Recombinant DNA
What two marker genes are often present in the plasmid in genetic engineering?
A fluorescent marker gene
An antibiotic resistant marker gene
How does the desired gene interact with the marker genes?
The gene will interrupt the fluorescent marker gene, meaning if the plasmid successfully takes up the gene, it will not fluoresce
What are the 3 methods of transferring the vector into the host cell in genetic engineering??
Heat shock
Electroporation
Electrofusion
What is heat shock?
A calcium rich solution and increased temperature causes the permeability of the host cell membrane to increase
What is electroporation?
When a small electric current in passed through the membrane of the host cell, making it very permeable
What is electrofusion?
When small electric currents are passed through the membranes of 2 different cells, fusing the two membranes
What is the product of electrofusion known as?
Polyploid cell
What other process uses electrofusion and how?
It is used in the production of monoclonal antibodies to fuse the antibody with the tumour cell
What happens to host cells that have no taken up any plasmid?
They will die in an antibiotic solution, as they have not taken up with antibiotic resistant marker gene
What happens to host cells that have taken up a plasmid which does not have the desired gene?
It will fluoresce as the desired gene has not interrupted the fluorescent marker gene
