common techniques Flashcards
What are some techniques used to study genes?
PCR
What is PCR?
A technique to amplify DNA fragments
What is needed for PCR?
DNA sample
What are primers in PCR?
Short DNA sequences complementary to the start of the target fragment.
What is DNA polymerase?
An enzyme that synthesizes new DNA strands.
Why is DNA polymerase important in PCR?
It withstands high temperatures without denaturing.
What is the first step of PCR?
Heat to 95°C to break hydrogen bonds and separate DNA strands.
What happens after DNA strands are separated in PCR?
The mixture is cooled to 50-65°C so primers can bind (anneal).
What is the next step after primer annealing in PCR?
Heat to 72°C to activate DNA polymerase.
How does DNA polymerase function in PCR?
It lines up free nucleotides to form complementary DNA strands.
What happens at the end of a PCR cycle?
Two new DNA copies are formed.
How does PCR amplify DNA?
Each cycle doubles the DNA amount (e.g.
Why is PCR useful?
It rapidly produces large amounts of DNA for analysis.
What is electrophoresis?
A technique that uses electrical current to separate DNA
What is the gel used in electrophoresis?
Agarose gel.
What is the purpose of buffer solution in electrophoresis?
It covers the gel and conducts electricity.
Where are the wells placed in the gel?
Near the negative electrode (cathode).
How are DNA samples prepared for electrophoresis?
They are mixed with loading dye to sink into the wells and make them visible.
Why is a micropipette used in electrophoresis?
To carefully load DNA samples into the wells without damaging them.
What happens after DNA samples are loaded into the wells?
An electric current is applied to the gel.
Which way do DNA fragments move in electrophoresis?
Towards the positive electrode (anode) because DNA is negatively charged.
How does DNA separate in electrophoresis?
Smaller fragments move faster and travel further through the gel.
How long does electrophoresis typically run?
About 30 minutes or until the dye is ~2 cm from the end of the gel.
What happens after electrophoresis is complete?
The gel is stained to visualize DNA fragments.
How is DNA fragment size measured?
In bases or base pairs (e.g.
Can electrophoresis be used for RNA?
Yes
How is protein electrophoresis different from DNA/RNA electrophoresis?
Proteins are denatured with a chemical to give them the same charge.
What is a use of protein electrophoresis?
Identifying proteins in urine or blood samples to diagnose diseases.
What are restriction enzymes?
Enzymes that recognize specific palindromic sequences and cut DNA at these sites.
What is a palindromic sequence in DNA?
A sequence that reads the same in opposite directions on complementary strands.
How do restriction enzymes recognize where to cut?
They bind to specific recognition sequences complementary to their active site.
Do all restriction enzymes cut at the same recognition sequence?
No
How can restriction enzymes be used to isolate a DNA fragment?
If recognition sequences flank the desired DNA fragment
What type of reaction do restriction enzymes use to cut DNA?
A hydrolysis reaction.
What are sticky ends in DNA fragments?
Small tails of unpaired bases left after a restriction enzyme cut.
Why are sticky ends useful in DNA manipulation?
They help DNA fragments anneal to other DNA with complementary sticky ends.