chap 3- DNA manipulation techniques and applications Flashcards
what are restriction endonucleases
enzymes that cut DNA at a specific recognition site
what are restriction enzymes used for
to cut DNA into smaller more usable fragments and isolate particular regions of interest (single genes)
When do restriction enzymes occur naturally
in bacteria
what do restriction enzymes form
bacterial cells defense system, targeting foreign DNA that may enter the cell
What are sticky-ends
when restriction enzymes leave DNA fragments with overhanging ends
what are blunt ends
when restriction enzymes leave clean-cut ends
how do sticky end restriction enzymes cut the DNA backbone
at a different location on each strand within the recognition site
how do blunt end restriction enzymes cut the DNA backbone
by cutting the sugar-phosphate backbone on both strands of the DNA molecule at the same location within the recognition site
What are ligases
a group of enzymes that join fragments of DNA or RNA and create a phosphodiester bond between them.
What are polymerases
enzymes that catalyse the formation of polymers, in particular nucleic acids
why does DNA polymerase act
to produce identical copies of DNA
what does RNA polymerase do
acts to assemble RNA. In cells, RNA polymerase synthesizes mRNA, rRNA and tRNA by transcription of genes.
what does PCR stand for
Polymerase Chain Reaction
what is PCR used for
to obtain larger amounts of DNA when there is only a small amount
In PCR: what does the DNA sample provide
a target sequence of DNA that is to be occupied and amplified.
In PCR what do primers provide
a starting point for DNA to be built on
what are primers
short nucleic acid sequences
In PCR what are free nucleotides used for
added to solution in order for DNA to be built
In PCR what does taq polymerase do
binds to primer and begins building DNA from the free nucleotides
In PCR what is the buffer solution for
provides suitable chemical environment for activity of polymerase
what is the significance of Taq polymerase for PCR
its extracted from bacteria that lives in hot springs meaning it can function in high temps without degrading
What are the 3 steps of PCR
denaturation. annealing. extension
what happens during denaturation
sample heated to 95 degrees. breaks hydrogen bonds between the strands of DNA to obtain single strands of DNA.
what happens during annealing
temp is reduced to 50-60 degrees. allows primers to bind to complementary base sequences through hydrogen bonds
what happens during extension
temp increased to 72 degrees. allows taq polymerase to attach to the primers on the DNA strands. the taq polymerase moves along each strand adding free nucleotides to form double stranded DNA.
in each PCR cycle how much is the number of double stranded copies of the DNA increased by
double the amount
what does gel electrophoresis do
separates fragments of negatively charged DNA by length
what can gel electrophoresis technique be used for
DNA screening, fingerprinting for forensics, paternity cases, confirming correct gene has been amplified by PCR
what does DNA profiling do
identifies one individual from another individual
what is DNA profiling used for
identifying perpetrators of a crime, identifier of bodies and to settle paternity disputes
what are non-coding DNA sections
regions that are not transcribed or translated into a protein