Biotechnology Flashcards
Purpose of Polymerase Chain Reaction
To amplify small amounts of DNA in a short period of time
Reactants added to the PCR tube:
- restricted s_____ of ___
- l_____ strand primer
- lagging s____ p____
- DNA p_________ (__q)
- Free de___________tides
- PCR t____, m_________, t__________
Reactants added to the PCR tube:
- restricted sample of DNA
- leading strand primer
- lagging strand primer
- DNA polymerase (Taq)
- Free deoxyribonucleotides
- PCR tubes, micropipette, thermocycler
Process:
Step 1: D________
Step 2: A_______
Step 3: E________
Process:
Step 1: Denaturing
Step 2: Annealing
Step 3: Elongation
Denaturing:
Temp:
Weak ________ _____ between _____ are _____, which results in two s_____ strands of ___ (ie strands are s_______)
Denaturing:
Temp: 94-96ºC (heating)
Weak Hydrogen bonds between bases are broken, which results in two single strands of DNA (ie strands are separated)
Annealing:
Temp:
P_____ (small single strand of DNA) anneals/b___ to the c___________ site on the l_____ and l_____ strands of ___ and starts the r________ process
Annealing:
Temp: 50-65ºC (cooling)
Primers (small single strand of DNA) anneal/bind to the complementary site on the leading and lagging strands of DNA and starts the replication process
Elongation: Temp: \_\_\_ \_\_\_\_\_\_\_\_\_ (DNA polymerase) r\_\_\_\_\_\_ segments of s\_\_\_\_ s\_\_\_\_\_ of \_\_\_ (ie add complementary sequence) using the free d\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ (dNTPs)
Elongation: Temp: 72ºC (heating) Taq Polymerase (DNA polymerase) replicate segments of single strands of DNA (ie add complementary sequence) using the free deoxyribonucleotides (dNTPs)
Taq Polymerase
A h___-_____ DNA p________ that does not b____ d___ when heated.
It is taken from a heat-_____ b______.
Taq Polymerase
A heat-stable DNA polymerase that does not break down when heated.
It is taken from a heat-loving bacterium.
DNA Profiling
After ___ has been used, an individual’s ___ is c__ into f_______ using r________ e______ at specific b___ s________. These f_______ are of varying l______ and are u____ to that individual.
DNA Profiling
After PCR has been used, an individual’s DNA is cut into fragments using restriction enzymes at specific base sequences. These fragments are of varying lengths and are unique to that individual.
Gel Electrophoresis
A method of s________ d______ p____ of ___ according to s___.
Prior to electrophoresis, ___ is c__ with r________ e______ at s______ s___ that d___ between individuals.
Gel Electrophoresis
A method of separating different pieces of DNA according to size.
Prior to electrophoresis, DNA is cut with restrictive enzymes at specific sites that differ between individuals.
Gel Electrophoresis
Step 1: DNA is l_____ into w___ at the _______ end of a ___ ___.
Gel Electrophoresis
Step 1: DNA is loaded into wells at the negative end of a gel bed.
Gel Electrophoresis
Step 2: An e_____ c____ is passed through the ___.
Gel Electrophoresis
Step 2: An electric current is passed through the gel.
Gel Electrophoresis
Step 3: The ________ charged ___ moves towards the ______ charge at the o______ end of the ___.
Gel Electrophoresis
Step 3: The negatively charged DNA moves towards the positive charge at the opposite end of the bed.
Gel Electrophoresis
Step 4: The ___ moves through the ___ at different s_____- ______ pieces move faster than _____ ones.
Gel Electrophoresis
Step 4: The DNA moves through the gel at different speeds- smaller pieces move faster than larger ones.
Gel Electrophoresis
Step 5: This forms b____ representing d_____ s___ s______ of ___. B____ may be called a ___ profile or ___ Fingerprint.
Gel Electrophoresis
Step 5: This forms bands representing different sized segments of DNA. Bands may be called a DNA profile or DNA Fingerprint.
