Lecture 5 Flashcards
What is hybridization probing and what can it be used for?
Hybridization probing can be used to identify recombinant DNA molecules contained in either bacterial colonies or bacteriophages plaques. It’s a fragment of DNA or RNA that can be radioactively or fluorescently labeled.
How do you do hybridization probing?
- The colonies/plaques are transferred to a nitrocellulose or nylon membrane
- The colonies are treated to remove all contaminating material, leaving just DNA. This treatment usually also results in the denaturation of the DNA –> the hydrogen bonds between individual strands in the double helix are broken.
- The single-stranded molecules can be bound tightly to the membrane by a short period of time at 80°C if nitrocellulose membrane is being used, or with a nylon membrane by UV irradiation.
- The molecules become attached to the membrane through their sugar-phosphate backbones so the bases are free to pair with complementary nucleic acid molecules.
- The probe must be labelled with a radioactive or other type of marker, denatured by heating and applied to the membrane in a solution of chemicals that promote nucleic acid hybridization.
- After letting the hybridization take place, the filter is washed to remove unbound probe, dried and the label detected in order to identify the colonies or plaques to which the probe has become bound.
What three kinds of stabilization modes are there?
- Unstable
- Stable
- DNA-RNA hybrid
Describe the different stabilization modes.
- Unstable. An unstable hybrid molecule is formed between two non-homologous DNA strands
- Stable. A stable hybrid molecule is formed between two complementary strands. Has short non-complementary regions that don’t affect overall stability.
- DNA-RNA. A DNA-RNA hybrid, like the ones that form between a gene and its transcript.
What two different markers can be used for labelling hybridization probes? Give examples.
- Radioactive labelling. Isotope 32P
2. Non-radioactive labelling. Luminol-Horseradish peroxidase, Avidin-Biotin interaction
What does the success of colony or plaque hybridization depend on? In regards of identifying a particular recombinant clone.
It depends on the availability of a DNA molecules that can be used as a probe. The probe must share at least a part of the sequence of the cloned gene.
If the gene is not available when doing hybridization probing, what can be used as the probe? In practice.
The nature of the probe is determined by the information available about the desired gene.
What three possibilities are there for choosing a probe in regard of the information available for about the desired gene?
- Where the desired gene is expressed at a high level in all cell type from which a cDNA clone library has been prepared.
- Where the amino acid sequence of the protein coded by the gene is completely or partially known.
- Where the equivalent gene from a related organism is available.
What is a genomic library?
A genomic library is a set of recombinant clones that contains all of the DNA present in an individual organism. An E. coli genomic library contains all the E. coli genes, so any desired gene can be withdrawn from the library and studied.
What is southern blot?
It’s a method to identify the individual restriction fragment containing a specific DNA sequence in DNA samples.
Describe the steps of southern blot.
- Restriction endonucleases are used to ut the DNA sample into smaller fragments.
- The fragments are electrophoresed on an agarose gel to separate them by size
- The DNA bands in the agarose gel are transferred to a nitrocellulose or nylon membrane, which provides a much cleaner environment for the hybridization experiment.
- The DNA fragments get to bind, hydridizate, to a DNA sequence which has been labelled to it can be detected, a probe
- Then you wash the probes that hasn’t bound to the DNA sequence on the membrane and detects the probe
- What you can see from the picture is where the probe has been, how large the piece has been and its strength is an indication of how common it is the DNA sequence.
What’s a genome?
A genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses).
How many bp of genome does humans and oat have?
Humans: 3 billion bp
Oat: 11-12 billion bp
What group is needed for chain elongation?
OH group
What is Sanger sequencing?
Deoxynucleotides (dNTP) are present in the DNA and is used to form the backbone of the DNA. dNTP has one OH-group that binds to the phosphate group of another dNTP which forms the backbone. If the OH-group is replaced with a H, it’s dideoxynucleotide (ddNTP). ddNTP can’t bind to phosphate groups which means that the polynucleotide chain reaction will stop when ddNTP is attached to the previous dNTP, since no other dNTP can bind to the ddNTP.
Classial sanger sequencing:
The template DNA that is to be sequenced is divided into 4 tubes. Each tube has a primer that will bind to the template DNa, all four dNTPs and DNA polymerase that will add dNTP on the template DNA. Each tube also has one radiolabelled dideoxynucleotide (at a very low concentration). The DNA polymerase add dNTPs as usual, but when it get to the ddNTP it will stop –> reaction terminates. The incorporation of ddNTP and termination is a random process –> the whole mixture will have several fragments that has experienced termination. If the length of the fragment is known, then the location of the ddNTP can be estimated. The length of the fragment can be optimized by electrogephoreses and autoradiogram.
Modern Sanger sequencing:
Each ddNTP is attached with a fluorescence dye. We can now have a gel that can give fluorescence when exposed to UV light. Only need one tube since each ddNTP has its own colour. The termination will happen if the ddNTPs are incorporated in the DNA. The fragments can be separated polyecrimongelelectrophoreses in a single well.
More advanced system:
Uses capillary electrophoresis for the separation of fluorescence DNA fragments. These capillary electrophoresis are equipped with CCD or charge-coupled device for the detection of fluorescence signals.
