Chapter Twenty-One: Manipulating genomes Flashcards
What is Polymerase Chain Reaction (PCR)?
- artificial DNA replication. it’s a technique that allows DNA fragments of interest to be copied many times (amplification)
- the amplified material can be used for many other genetic techniques
What are the reagents for PCR?
- DNA
- primers
- DNA nucleotides
- DNA polymerase (Taq)
What happens in the first stage of PCR?
- the DNA sample is mixed with primers, DNA nucleotides, and Taq (DNA polymerase)
- the vial is placed in a PCR machine
What happens in the second stage of PCR?
- the mixture is heated to 95°c, breaking the hydrogen bonds that hold the complementary strands together
- the double stranded DNA sample denatures to make simple stranded
DNA - DNA polymerase dose to denature since it’s a ‘thermophilic’ version (Taq)
What happens in the third stage of PCR?
- mixture is cooled to around 55°c, allowing the primers to anneal
- the primers (short sequences of nucleotide bases) must join to the start of the separated DNA strands so the full copying process can start. Taq is able to bind to these double stranded areas
What happens in the fourth stage of PCR?
- Mixture is heated up t0 72°C as this is the optimum temperature for the DNA polymerase enzyme
- the DNA polymerase extends the small sections of double stranded DNA by adding free nucleotides to the unwound DNA
What happens in the fifth stage of PCR?
- two new copies of the fragment of DNA are formed and one cycle of PCR is completed
- the cycle starts again
What are three advantages of PCR?
- automated process making it more efficient
- rapid process, 100 billion copies can be made in a few hours
- it doesn’t require living cells making it quicker and less complex techniques are needed
What are restriction enzymes?
- another way to get a DNA fragment from an organisms DNA
What are palindromic sequences?
- sequences that consist of antiparallel base pairs (base pairs that read the same in opposite directions)
What do restriction enzymes do?
- they recognise specific palindromic sequences (recognition sequences) and cut (digest) the DNA at these places
Why do different restriction enzymes cut at different specific recognition sequences?
- because the shape of the recognition sequence is complementary to an enzymes active site
How to use restriction enzymes to separate the DNA fragment you want that’s in between recognition sequences?
- DNA sample is incubated with the specific restriction enzyme, this cuts the DNA fragment via a hydrolysis reaction
- the cut can leave sticky ends (small tails of unpaired bases at each end of the fragment) that can be used to anneal the DNA fragment to another piece of DNA that has sticky ends with complementary sequences
What does gel electrophoresis do?
- it uses an electrical current to separate out: different fragments of DNA, RNA fragments, and proteins according to their sizes
What happens in the first step of gel electrophoresis?
- DNA fragments are treated with restriction enzymes to cut up the fragments and the DNA samples are placed unto the wells cut in one end of the gel (closest to the negative electrode)
What happens in the second step of gel electrophoresis?
- the gel is put in a tank of buffer solution and an electric current is passed through the solution for a fixed amount of time (usually around 30 mins to 2 hrs)
- DNA is attracted to the positive electrode as it is negatively charged
- DNA fragments diffuse through the gel
True or false?:
1. longer lengths of DNA move faster than shorter lengths
2. the position of DNA fragments can be shown by dye stains
3. fragments can’t be lifted from the gel
- false - shorter lengths travel faster
- true
- false - they can be lifted from the gel for further analysis (southern blotting)
What happens in the third step of gel electrophoresis?
- a nylon sheet is placed over the gel, covered in paper towels, pressed and left over night
- DNA fragments are transferred to the sheet and can be analysed
electrophoresis can be carried out on RNA fragments using the same method as DNA fragments, but what is the difference between them?
- because proteins can be positively or negatively charged, they are mixed with a chemical that denatures the proteins so they all have the same charge before undergoing electrophoresis
define the following words: genome, exons, introns, satellite DNA, minisatellite/VNTR’s, microsatelite/STR’s.
- Genome – all the genetic material an organism contains
- Exons – DNA which codes for protein (genes)
- Introns – regions of non-coding DNA
- Satellite DNA – short sequences of DNA that are repeated many times
- Minisatellite/VNTR’s – sequence of 20-50 base pairs that are repeated 50-100s of times
- Microsatellite/STR’s – 2-4 bases repeated only 5-15 times
define DNA profiling
- producing an image of the patterns in a persons DNA, creating a profile that is individual to them
what are some uses of DNA profiling?
- forensics: DNA found at a crime scene can be compared to victims or suspects
- proving paternity of a child
- immigration cases to provide family relationships
- identifying individuals who are at risk of developing a disease
- evolutionary relationships between different species
what substances are needed to make a DNA profile?
- DNA from a tissue sample
- PCR machine
- restriction endonucleases
- gel electrophoresis machine
- radioactive/flourescent DNA probes
- x-ray machine/UV light
what are the six main steps of creating a DNA profile?
- extract DNA from samples
- increase amount of DNA using PCR
- cut sample up using restriction endonucleases
- separate DNA fragments using electrophoresis, then southern blotting
- hybridisation- radioactive/fluorescent DNA probes added which binds to the complementary strands of DNA and identify the microsatellite area
- seeing the evidence: x-rays taken/UV light to show fluorescent tags
define genetic engineering. what is another name for genetic engineering?
- combining DNA from different organisms or from different sources
- recombinant DNA technology because recombinant DNA is formed which is a section of DNA that has been made from two different sources
fill in the words for this gap fill:
- genetic engineering is the m____________ of an organism’s DNA
- Organisms that have had their DNA altered are called t_________ or transformed organisms/genetically modified
- They have r___________ DNA (a section of DNA that has been made from two different sources), so it is also called recombinant DNA technology
- It involves extracting a gene from 1 organism and inserting it into another organism DNA (often of another species)
- Genes can also be m_____________ instead of extracted
- The organism with the new gene will then be able to produce the p_________ coded for by that gene
- genetic engineering is the manipulation of an organism’s DNA
- Organisms that have had their DNA altered are called transgenic or transformed organisms/genetically modified
- They have recombinant DNA (a section of DNA that has been made from two different sources), so it is also called recombinant DNA technology
- It involves extracting a gene from 1 organism and inserting it into another organism DNA (often of another species)
- Genes can also be manufactured instead of extracted
- The organism with the new gene will then be able to produce the protein coded for by that gene
what are the four stages of genetic engineering?
- obtaining/isolating the desired gene
- making the recombinant DNA
- transferring the vector (transformation)
- identify the transformed bacteria
in step 1 of genetic engineering, the desired gene must be cut out of the DNA where it’s found. what are the two ways of doing this? describe them
- restriction enzymes: cut through DNA at specific points producing sticky or blunt ends.
(normally come from bacterial cells, there’s over 50 different restriction enzymes) - isolating the mRNA for the desired, then using reverse transcriptase to produce a single strand of the complementary DNA (cDNA)
- this is what’s done to make insulin, the mRNA from the beta cells is extracted
in step two of genetic engineering, the DNA fragment is inserted into a vector. what is a vector? what are the three steps of making the recombinant DNA?
- vector: something that is used to transfer DNA into a cell. can be plasmids or bacteriophages
- vector DNA is isolated
- vector DNA cut open using the same restriction enzyme used to isolate the DNA w/ the desired gene, so the sticky ends of the vector DNA is complementary to the DNA fragment with the gene
- vector DNA and DNA fragment are mixed together with DNA ligase, joining the sugar phosphate backbones of the two bits of DNA. recombinant DNA is now formed
- ligation = forming phosphodiester bonds
in step three of genetic engineering, the vector with the recombinant DNA transfers/carries the gene into the host cell (bacterium). in most cases the vector is a plasmid and the bacterium has to be encouraged/persuaded to take it up. what are the three ways to get the bacterium to do this?
- electroporation: suspension of bacterial cells and plasmid vector is put in an electroporator. a small current is applied to the bacteria, increasing the permeability of their cell membranes so the plasmids can move in
- culture the bacterial cells and plasmids in a calcium-rich solution and increase the temp. this makes the bacterial cells membrane more permeable so plasmids can enter
- electrofusion: tiny current is applied to the membranes of the two different cells which fuses the cell and nuclear membranes of the two different cells. this forms a hybrid/polyploidy cell that has DNA from both
if the vector in genetic engineering is a bacteriophage, how is the recombinant DNA transferred into the host bacterium?
- it injects its DNA into the host bacterium and the phage DNA with the desired gene integrates with the bacterial DNA
in step four of genetic engineering, not all bacteria will have taken up the plasmid. you only want to culture and grow the ones you have.
what can be used to identify the bacteria that has taken up the gene?
marker genes
