Flashcards in 1A- Intro to DNA Technology Deck (53)
What are some advantages that technology offers with DNA?
Cut, modify, reassemble and analyze DNA
Select, characterize, mutate in a predetermined manner and even synthesize individual genes;
Clone and express individual genes to produce the proteins they encode in large amounts (e.g. insulin, human growth factor);
Determine and locate the gene mutations responsible for genetic diseases;
Diagnose sufferers and carriers of genetic diseases;
Plan future therapies that involve manipulation or replacement of genes.
What is a restriction endonuclease?
It is an enzyme that cuts DNA at specific nucleotide sequences (restriction sites).
How does a restriction endonuclease cut DNA?
To cut the DNA, they need to make 2 incisions, once through each of the sugar-phosphate backbone of the DNA
What are recognition sites?
They are locations on DNA that contain specific sequences of nucleotides, which are recognized by restriction enzymes.
In what form does restriction sites usually come in?
They are typically palindromic, which means both strand of the DNA have the same base sequence when read in a 5’-3’ direction.
What is a blunt end?
the cut DNA is double-stranded at both ends, with no overhangs.
What are sticky ends?
the cut products are single-stranded at the ends, with 1 strand overhanging the other.
What is DNA ligase?
an enzyme that joins DNA strands together by forming a phosphodiester bond
What is a linker?
a piece of DNA that is used to put on a blunt end of a DNA fragment which has been cut by restriction endonucleases.
What is DNA polymerase?
It is an enzyme that synthesizes DNA in a 5’-3’ direction from a DNA template
What is reverse transcriptase?
Reverse transcriptase is an enzyme that synthesizes DNA from an RNA template.
What type of organisms use reverse transcriptase to make DNA?
You can use reverse transcriptase in bioengineering on mRNA to produce what?
What does cDNA not have that regular DNA has?
Introns and regulatory regions of the gene. Remember this is coming from mature mRNA, where all the introns are spliced out.
What is hybridization?
It is the process of binding 2 complementary strands of nucleic acids together
What is the term of the temperature at which 50% of the DNA is separated?
What is a probe?
A probe is a single-stranded DNA or RNA that is used to identify a complementary sequence on a larger single-stranded DNA or RNA
What molecule does western blotting detect?
What does western blotting use as a probe for the proteins?
Give the process of a western blot to identify a specific protein
proteins added to gel --> gel electrophoresis to separate based on size --> blot to nitrocellulose membrane --> add radioactively-labelled antibodies --> wash --> autoradiography to see binding.
What type of molecule does a Southern blot detect?
What type of probe does a southern blot use to detect DNA?
Give the process of a southern blot to find a specific DNA sequence
restriction endonucleases cut DNA --> electrophoresis of fragments --> alkaline solution to denature dsDNA --> nitrocellulose membrane to transfer DNA onto it --> bake with UV --> use radioactive DNA probe --> wash --> autoradiographic analysis
What type of molecule does Northern blots detect?
What type of probe does northern blots use to detect RNA?
What does an RFLP test for?
variations in the lengths of restriction fragments
Why would there be variations in lengths of restriction fragments?
There could be a mutation in a recognition site so the endonuclease doesnt cut, making the fragment longer and less in #. Or, there could be a mutation in the genome to make a recognition site, making the fragments shorter and more in #.
What is a VNTR?
VNTR's are sections of the DNA that contain a repeated tandem # of sequences for a variable # of times
What is significant of VNTR's between different people?
They differ in size