Wha happens when double stranded DNA is heated or treated with alkaline solution?
It denatures which breaks the hydrogen bonds and releases single stranded DNA (ssDNA)
What is the basis for DNA hybridisation?
- dsDNA is heated so that the hydrogen bonds are broken and it is denatured.
- Then, before cooling, complimentary ssDNA with a radioactive or fluorescent marker is added.
- some of the original ssDNA will anneal with the labelled DNA
- This labelled DNA can now be identified using photographic film.
What are the differences between Southern, northern and western blotting?
- Southern blotting was invented by (and named after) after Prof Sir Ed Southern in 1975 and to uses probes to identify complimentary DNA sequences after gel electrophoresis.
- Northern blotting was invented in the late 1970s (not named after a person) and it used DNA to detect RNA species in a similar way to southern blotting.
- Western blotting is NOT a DNA hybridisation technique. It uses similar principles to detect proteins using antibodies after protein gel electrophoresis.
What are the stages of Southern blotting?
- Digest DNA using restriction enzymes,
- Use DNA gel electrophoresis to separate the fragments of DNA,
- Transfer DNA fragments to a nylon or nitrocellulose membrane and soaked in alkali to denature the DNA,
- Hybridise filter with a labelled gene probe to detect a specific piece of DNA,
- Wash the filter to remove any unbound probe and detect hybridisation (and therefore DNA of interest) by exposing the filter to an X-Ray film.
Why do we use Southern blotting?
- To investigate gene structure. Eg large deletions of duplications
- To investigate gene explanation such as triplet repeats. Eg Fragile X syndrome and Huntington’s,
- To investigate mutations in genetic tests - this uses allele specific probes eg for Sickle Cell disease,
- To investigate variation and genetic relationships eg DNA fingerprinting.
What other technique is Southern blotting often used in combination with and why?
PCR to allow easier identification as PCR amplifies the gene. This means that Southern blotting allows us to detect very small amounts of DNA that may not be visible by staining of DNA in a gel.
What two things do probes not have to be?
Probes do not have to have 100% similarity to the target sequence. 80% would be enough. All this means is that it would bind less tightly.
Probes also do not have to completely align with the target sequence. Even if it only binds to part of the sequence, it is enough to allow detection.
What do probes not affect?
Probes do not affect the position of a target sequence on a gel. This is because the number of base pairs and therefore length of the sequence will be the same.
What is the purpose of the Sanger chain method?
It is also known as the dideoxy chain termination method. It allows us to work out the nucleotide sequence of DNA depending on when termination occurs.
What is a ddNTP
A ddNTP is a dideoxynucleotide triphosphate. It is the same as a normal DNTP (base) which is usually used in DNA replication except, it has a H rather than an OH at 3’. This means that, if this binds, DNA replication cannot continue because there is no OH to form the phosphodiester bond with the next base.
What is the Sanger chain termination method?
- In this method, 4 separate test tubes are used. Each with a different ddNTP in (A,C,T,G), the DNA and primers to initiate DNA replication.
- These test tubes are incubated at 37°C and DNA left to replicate.
- After incubation, the products of the reaction are run out using separate lanes for each of the reaction test tubes. This will separate the labelled fragments out on the basis of size.
- We are then able to read off the sequence from the bottom of the gel to work out the nucleotide sequence in a newly synthesised strand.
What variation on the Sanger chain termination method is used now?
Now, we use fluorescently labelled ddNTPs all in the same tube. The different length fragments are then separated on a very thin capillary. As the fragments fall off the end, they are detected by a laser. This is then produced as a chromatogram where read the sequence directly.
What restriction endonucleases? What are they used for?
They are usually very specific palindromic sequences (same forwards as backwards) produced by bacteria. They:
- Recognise and degrade foreign DNA
- Recognise and cut specific DNA sequences
- They are ‘MOLECULAR SCISSORS’
What are the four requirements for DNA gel electrophoresis?
- Gel -A matrix that allows for the separation of DNA fragments
- Buffer- Allows charge on the DNA samples across the gel
- Power Supply - This generates a charge difference across the cell
- Stain/Detection -eg ethidium bromide to allow the identification of the presence of the separated DNA.
Why can gel electrophoresis be used to separate DNA molecules of different sizes?
Because DNA is negatively charged (the phosphate) and so will move towards the positive electrode (the anode) is placed in an electric field. The larger fragments move slowly and smaller fragments move faster which then allows the separation to occur.
Why do we use restriction analysis?
- To investigate the size of DNA fragments eg small deletions
- To investigate mutations eg sickle cell disease
- To investigate DNA variation eg DNA fingerprinting
- To clone DNA
What are plasmids?
They are small, circular dsDNA that are found in bacteria. The are like “mini chromosomes” as they carry genes to replicate independently. They also often carry antibiotic resistant genes and can be transferred to other bacteria.
What are the four basic steps of gene cloning?
- Isolate the relevant gene of interest following PCR and digestion with restriction enzymes
- Insert gene of interest into a plasmid vector (to create a recombinant DNA molecule)
- Introduce the recombinant DNA molecule into suitable host cells eg E. coli
- Identify and isolate the clone containing the gene of interest.
Why do we clone human genes?
To make useful proteins eg insulin
To find out what genes do or the difference between individuals
Genetic screening eg for Huntington’s, BRACA1/2, Cystic Fibrosis
Gene therapy eg cystic fibrosis (get wild type (normal) gene into patient using inhalers)
What is PCR?
PCR is the polymerase chain reaction and it is used to amplify target DNA.
Describe the process of PCR
- Heat the DNA to 95°C to DENATURE it (break the H bonds).
- Cool to 60°C to allow both the specific forwards and reverse primers (sequence of Oligonucleotides) to ANNEAL to the DNA strands.
- Heat to 72°C which is the optimum temperature for Taq Polymerase to add new nucleotides to the strand (POLYMERISE).
- Repeat 20-35 times. This results in an exponential increase in DNA.
Why do we use PCR?
To amplify a specific DNA fragment
To investigate specific single base mutations eg Tay Sachs and Sickle Cell
To investigate small deletions or insertions eg Cystic Fibrosis
To investigate variation and genetic relationships eg DNA profiling or DNA typing or DNA fingerprinting.
What are the differences between protein and DNA gel electrophoresis?
- Proteins can be either positive or negatively charged so can move towards the anode or the cathode. DNA always moves towards the anode as it is negatively charged.
- Protein electrophoresis is conducted vertically whereas the DNA electrophoresis is conducted horizontally.
- In protein, it is quantitative so you can different intensities of colour or peaks on a graph to show the level of protein (eg in blood serum).
- Protein electrophoresis is most commonly used to diagnose multiple myeloma
What is isoelectric focusing?
IEF is when proteins separate on the basis of charge. Proteins migrate in a gel with a stable pH gradient (created using an electric field) until they reach a neutral pH equal to their pI. They they reach this point, there is no net charge so they stop migrating.
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Enzyme Activity12
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Chromosomal Abnormalities24
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Mutations (Prof London)32
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Random1
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Analysis Techniques56
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Regulation Of Protein Function31
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Oxygen Transport23
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Mutations17
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Post Translational Processing Of Proteins And Protein Targeting45
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Genotype, Phenotype And Inheritance13
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Protein Folding- Review Of Protein Structure9
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Genetics in Medicine (History)14
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Nutrition, Diet and Energy Metabolism60
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Energy Reactions In Cells38
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Energy Production: Carbohydrates86
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Energy Production: Lipds27
