Flashcards in Locating And Sequencing Genes Deck (17):
What are the two most commonly used probes?
Radioactively labelled probes, fluorescently labelled probes
What is a DNA probe?
A short, single stranded section of DNA that has some sort of label attached that makes it easily identifiable
What are radioactively labelled probes made of and how are they identified?
They are made using nucleotides with the isotope and is identified using a photographic plate that is exposed by radioactivity
How are fluorescently labelled probes identified?
They emit light under certain conditions
How are DNA probes used to identify particular genes?
A DNA probe is made that has bases that are complementary to the portion of the DNA sequence that makes up part of the gene whose position we want to find. The DNA that is being tested is treated to separate its two strands which are mixed with the probe which binds to the complementary bases (DNA Hybridisation). The site at which the probe binds can be identified by the radioactivity or the fluorescence that the probe emits
What must be done before we can make a specific probe?
We need to know the sequence of nucleotides in the particular gene that we are trying to locate
Name a method that is used to sequence the exact order of nucleotides in a section of DNA
The Sanger method
What does the Sanger method use?
Modified nucleotides that cannot attach to the next base in the sequence when they are being joined together
What do the modified nucleotides act as?
Terminators that end the synthesis of a DNA strand
What is the first stage of the Sanger method?
Set up 4 test tubes containing many single stranded fragments of the DNA to be sequenced, a mixture of nucleotides (ATGC), a small quantity of one of the 4 terminator nucleotides, a primer to start the process of DNA synthesis and DNA polymerase
What will determine when the DNA synthesis terminates?
Where the terminator nucleotide binds to the DNA template
How will the DNA fragments be able to be identified?
Because the primer attached to the other end of the DNA section is labelled radioactively or with a fluorescent dye
What is the next stage of the Sanger method?
The different length fragments of DNA are separated
How are the different length fragments of DNA separated?
What is involved in gel electrophoresis?
The DNA fragments are placed on to an agar gel and a voltage is applied across it
How does the resistance of the gel affect the movement of the DNA fragments?
The larger fragments move more slowly whereas the smaller fragments move further which causes the DNA fragments to separate