Genetics Flashcards
What are the five stages of DNA technology and what is involved in these five stages?
Isolation- of the required gene
Insertion- of the DNA into a vector
Transformation- Transfer DNA to a suitable host
Identification-Finding the host organisms that contain the vector and DNA
Growth/Cloning of the successful host cells
What does reverse transcriptase do?
It converts mRNA to cDNA (does the opposite of transcription).
What is the role of DNA polymerase when used in conjunction with reverse transcriptase?
After the cDNA is produced, the single strand is binded to with other DNA nucleotides by DNA polymerase, creating a double helix of cDNA.
Why do bacteria contain restriction enzymes?
To stop viruses from invading them by cutting their DNA
How do restriction enzymes form sticky ends?
They make a staggered cut to the DNA
What do two DNA strands need to be able to join at their “sticky ends”?
To be cut by the same restriction enzymes.
What are recognition sites?
specific sites on DNA, usually 4-8 bases long. The sequence on two DNA sites must be the same but backwards (palindromic) in order to bind
What enzyme joins sticky ends?
DNA ligase
What are organisms that contain recombinant DNA known as?
either a transgenic or genetically modified organism
Describe the process of the gene machine?
- The desired sequence of DNA is fed into a computer and checked against biosafety/biosecurity standards
- The computer designs a series of oligonucleotides which are joined to make the desired gene
- The DNA strand is replicated to form a double strand using PCR
- This gene can then be inserted into the bacterial plasmid using sticky ends (the bacteria is a vector)
What are oligonucleotides?
They are created in the gene machine. They are a series of small overlapping ingle strands of nucleotides
What are four advantages of the gene machine?
- Quick
- Accurate
- No introns or other pieces of non-coding DNA
- Easily transcribed in prokaryotic cells
What is the point of PCR?
To multiply the desired gene frequent enough times that it is usable.
What are the number of carbons that relate to DNA strands?
On one strand of DNA the 5’ carbon is at the top of the strand while the 3’ carbon is at the bottom of the strand. On the other strand, the nucleotides are inverted so the 3’ carbon is at the top of the strand while the 5’ carbon is at the bottom of the strand.
In what direction does DNA polymerase travel in DNA replication?
From the 3’ end to the 5’ end.
What are DNA primers?
Small sections that bind to the correct strand and cause DNA polymerase to bind to and copy the right strand.
In brief summary, what are the three stages in the polymerase chain reaction?
FIrst, the DNA strands are heated to 95 degrees to denature the two bases and split the two DNA strands. The strands are then cooled to 55 degrees when the primers are added to allow them to bind and so the DNA polymerase can work without being denatured. The strands are then cooled to 72 degrees as new nucleotides are added to create the new DNA strands.
What is PCR (as it occurs in a lab)?
In Vitro
What are three uses of PCR?
- For paternity tests- to produce enough DNA so one can compare the DNA of the kids to the DNA of the father.
- To see/test if there is a mutation of a specific gene
- In forensics to test and duplicate DNA samples
What is a thermocycler?
A computer machine that varies temperature for set time periods.
What is the promoter region?
It is a region near a gene that RNA and DNA polymerase bind to.
what is the terminator region and what must it ensure?
It stops transcription by causing RNA/DNA polymerase to detach.
What happens when restriction enzymes cut open a plasmid?
It disrupts one of the antibiotic resistance genes while the other antibiotic resistant gene is used in order to identify which vectors contain the recombinant DNA.
How are calcium ions important in reintroducing the recombinant plasmid to the vector bacteria?
The plasmids are reintroduced to the bacteria in a medium containing calcium ions. This causes the bacterial membrane to become permeable, therefore allowing the plasmids to pass into the cytoplasm.
Why do not all of the bacteria in a sample end up with the desired, recombinant gene?
(three points)
- Because only a few of the bacterial cells take up the plasmids when the two are mixed together
- Because some plasmids will have closed up again without incorporating the DNA fragment
- Because sometimes the DNA fragment ends can join together to form their own plasmid
How can we use antibiotics to identify cells with the desired recombinant DNA?
The bacteria are grown on a medium containing antibiotics. When the recombinant gene is inserted into the plasmid it is inserted into an antibiotic resistance gene (there are two, tetracycline resistance gene is affected) so this antibiotic resistance gene will not function anymore. Therefore the bacteria that don’t grow in the medium contain the desired DNA.
How can fluorescent markers be used to identify cells with the desired recombinant DNA?
The gene from gene from jellyfish that produces green fluorescent protein (GFP) is incorporated into the plasmid. The desired gene is inserted into the centre of the GFP gene, disrupting the production of GFP. If the DNA fragment is taken up, the bacteria will not glow and can be identified.
How can lactose be used to identify cells with the desired recombinant DNA?
The enzyme lactose will turn a colourless substance a blue colour. The DNA fragment can be transplanted into the gene that makes lactose, therefore disrupting it. If taken up, when the plasmid is mixed with a colourless substrate, it will remain colourless.
What are core sequences?
The sequences of introns in DNA
what happens in stage 1 of gene fingerprinting?
DNA is extracted from a sample (i.e. a strand of hair, saliva). The person will usually then carry out PCR. The DNA is usually cut into restriction fragments by restriction enzymes. These enzymes end up being all different lengths, some long, some short.
What happens in stage 2 of gene fingerprinting?
gel electrophoresis
Fragments are separated by their size using a process called gel electrophoresis. This is when a gel container is exposed to an electrical current by an anode and a cathode. The DNA fragments are negatively charged, so they travel to the anode. However, as the fragments are different sizes, they travel at different speeds. Therefore, the lighter/shorter fragments end up closer to the anode.
What happens in stage 3 of gene fingerprinting?
Southern Blotting
The gel containing the DNA fragments is immersed into an alkali in order to separate the double strands into single strands. The pattern of fragments are then transferred to a nylon membrane by a process called southern blotting.
What happens in Southern Blotting?
A thin nylon membrane is laid over the gel containing DNA fragments
The membrane is covered in absorbent paper which draw up the liquid containing the DNA
This transfers the DNA to the nylon membrane in the same positions as they were in the gel. The fragments are then fixed to the membrane using UV light.
What happens in stage 4 of gene fingerprinting?
probes
Radioactive probes are used to attach to the core sequence
The probes have base sequences that are complementary to the core sequences
Any probes not bound are washed off
The membrane is the dried
What happens in stage 5 of gene fingerprinting?
x ray film
The nylon sheet is placed under x-ray film
The radioactive probes expose the film
This produces patterns of film and dark bands which are unique to each individual
The pattern is then analyzed
How can gene fingerprinting be used in crimes?
The pattern of the DNA profile can be compared to that of the victim and the suspect. If the profile matches the suspect it provides evidence that the suspect was present at the crime scene.
How does gene profiling relate to population variability?
A population whose members have very similar genetic fingerprints has little genetic diversity, while a population whose members have a greater variety of genetic fingerprints has greater genetic diversity.
What are two advantages of In Vitro gene cloning?
It is rapid
It does not require living cells, all that is needed is the base sequence that needs amplifying.
What are four advantages of In Vivo cloning?
There is no risk of contamination, as the DNA is cut by restriction endonucleases so contaminant DNA can not bind to the recognition sites
It is accurate, with few errors
Can cut and use specific genes
Can produce large quantities of gene products, which can be used for commercial or medical use
What are five points supporting recombinant DNA technology?
This technology can be used to produce beneficial substances such as insulin
Genetically modified plants can produce useful substances
Genetically modified crops can have financial and environmental advantages
Relacing DNA can lead to cures for disease
Genetic fingerprinting can be used in paternal identification and forensic science.
What are the non-coding parts of DNA called?
variable number tandem repeats
what are six points against recombinant DNA technology?
Manipulation of DNA will have consequences for metabolic pathways.
Genetically modified bacteria may spread antibiotic resistance genes.
The gene may evolve to be adapted into the rest of the population
There are financial issues
If this technology gets into the wrong hands, there would be serious consequences.
Some could argue that it is immoral
How can tumor suppressor genes cause cancer?
If the TSGs are suppressed they can cause uncontrolled cell growth. This can occur due to hypermethylation of the gene. This will lead to tumor suppressors not being produced.
What is apoptosis?
Programmed cell death
What are tumor suppressor genes?
They are genes that make proteins that slow down cell division, repair mistakes in DNA and trigger Apoptosis
How can oncogenes cause cancer (2 ways)
They can become permanently switched on, resulting in excess cell division. They do this either by-
- causing the receptor proteins on cells to remain constantly switched on and not require growth factors
- cause excessive production of growth factors