Simple molecular techniques Flashcards
What are restriction enzymes?
Endonucleases that cut specific DNA sequences
What are the specific DNA sequences that restriction enzymes cut called?
Restriction sites
What are some common features of restriction sites?
Less than 10bp long
Palindromes
What are palindromes, in the context of DNA?
Both strands of DNA read the same base sequence in opposite directions
What are the types of cuts produced by restriction enzymes?
Blunt ends
Sticky ends
What is smeant by sticky ends?
Staggered cut
single-stranded overhangs
What is the purpose of DNA gel electrophoresis?
To separate out DNA fragments
DNA gel electrophoresis separates out DNA fragments based on their…?
Size
What are the requirements of DNA gel electrophoresis?
Gel
Buffer
Power supply
Staining
What is the purpose of the gel in DNA gel electrophoresis?
To give resistance to DNA fragments
What is the purpose of the buffer in DNA gel electrophoresis?
Maintain charge on DNA fragments
What is the purpose of the power supply in DNA gel electrophoresis?
Generate charge difference across gel
What is the purpose of staining in DNA gel electrophoresis?
So the DNA fragments are visible and can be identified
Why do the DNA fragments move across the gel in DNA gel electrophoresis?
DNA is negatively charged
will move towards the positive end of the gel
Why do DNA fragments separate out when they are moving across the gel in DNA gel electrophoresis?
DNA fragments are different sizes, weights
heavier DNA fragments face more resistance from gel
move slower across gel
How are the results of DNA gel electrophoresis interpreted?
Comparing position of unknown lengths of DNA
to positions of known lengths of DNA
What is gene cloning?
Producing copies of specific genes and their products
How are restriction enzymes used in gene cloning?
Used to isolate the gene from the rest of DNA
Used to cut the plasmid open
How are plasmids used in gene cloning?
Gene is inserted into plasmid
How is the specific gene inserted into the plasmid in gene cloning?
Because the gene sticky eneds are complementary to the plasmid sticky ends
base pairs form
DNA ligase anneals the two together
What is done to the recombinant plasmid in gene cloning?
Mixed with bacteria
some bacteria take it up - called transformation
What type of gene do plasmids commonly contain?
Antibiotic resistance genes
How are transformed bacteria told apart from non-transformed bacteria in gene cloning?
Only transformed bacteria will have specific antibiotic resistance gene
only these bacteria will survive in environment of that antibiotic
What is the purpose of PCR?
To amplify DNA fragments
What are the requirements of PCR?
DNA fragment to be copied
Primers - forward and reverse
Free dNTPs
Taq polymerase
Thermocycler
What are primers?
Short single-stranded sections of DNA
What are the stages of PCR?
Denature DNA
Add primers
Elongation