Nucleic Acid Probes Flashcards
what is a genomic library?
a set of recombinant clones, each carrying copies of a particular fragment from the genome
what kind of bacteriophage vector is most commonly used?
bacteriophage lambda
why is bacteriophage lambda used?
well known genome, can hold larger amounts of DNA, plaques form where the bacteria have been lysed
what is BAC?
bacterial artificial chromosome that ca =n be used as a vector
how is BAC useful?
can carry a large DNA insert so minimises the number of clones needed to make a genomic library
how can a more limited gene library be created?
by making a cDNA library
how is a cDNA library made?
begin with mRNA and use reverse transcriptase to create cDNA
how is cDNA made?
reverse transcriptase makes the first DNA strand, the mRNA is degraded and DNA polymerase makes the second strand
what primer is used by reverse transcriptase to make the first strand?
poly-dT
why are cDNA libraries more useful?
as they contain the complete coding sequence of the DNA but with no introns
in what situations are cDNA librarys used?
if only interested in the coding sequence of the DNA or studying gene expression of different stages in development
how are nucleic acid probes used?
DNA made single stranded, hybridised on a filter on which the probes are incubated, and then tested for radiography
how can nucleic acid probes then identify transformed colonies?
the filter can be compared to the master plate
what does electrophoresis do to the DNA fragments?
sorts them into bands of the same base pair length
why do longer fragments travel less far than the shorter fragments?
the polymer fibres in the gel impede the longer fragments more than the shorter ones
how can you compare two different alleles?
using restriction enzymes as they will differ in their sequence and so have different restriction sites so different band patterns
what does southern blotting do?
allows detection and analysis of particular sequences using nucleic acid hybridisation
how is DNA prepared for the blotting process?
digested with restriction enzymes and separated using electrophoresis
how does southern blotting take place?
an alkaline solution is pulled up through the gel and through a sheet of nitrocellulose paper on top of the gel
how does the blotting process allow analysis of the DNA?
as the DNA is transferred to the paper and made single stranded, then treated with probes and tested for hybridisation
what are RFLP’s?
restriction fragment length polymorphisms, differences among individuals in the lengths of DNA fragments cut by enzymes
how can RFLP’s be useful?
act as a genetic marker for mutations
