DNA TECHNOLOGY L17 Flashcards
I. PCR - SEQ, HD PCR, gel electrophoresis II. DNA sequencing - SEQ, HD dideoxy sequencing III. Applications - HD, APPLY DNA technology
1
Q
PCR
A
- method for producing many copies of desired DNA in short time
- important when studying genes
2
Q
template DNA
A
- sample containing the DNA sequence you want to amplify
3
Q
dNTPs
A
- deoxyribonucleotides
- A,T,C,G
4
Q
primers
A
- short DNA fragments with specific sequences at each end of your target DNA sequence
5
Q
taq polymerase
A
- DNA polymerase
6
Q
process of PCR
A
- denaturation
- annealing
- extension
7
Q
denaturation
A
- heat to 90
- breaks H bonds in double helix
8
Q
annealing
A
- 40-65
- primers bind with complementary sequences in template strand
9
Q
extension
A
- 72
- taq polymerase replicates DNA from 3’ end of primers
10
Q
gel electrophoresis
A
- often used to confirm PCR results
- separates nucleic acids or proteins based on size
11
Q
agarose gel
A
- polysaccharide gel
- pass electrical current through gel
- DNA moves from - to +
12
Q
dideoxyribonucleotide chain termination sequencing ingredients
A
- target DNA: what you want to sequence
- primer: short DNA molecule, designed to base pair with 3’ end of template strand
- DNA polymerase
- 4 deoxynucleotides
- 4 dideoxynucleotides
13
Q
dideoxyribonucleotide chain termination sequencing process
A
- target DNA denatured
- complementary strands synthesized
- dideoxyribonucleotides randomly incorporated
- elongation stops at dideoxy nucleotide
14
Q
dideoxyribonucleotide chain termination sequencing results
A
- many complimentary strands synthesized
- all different lengths
- each labeled with fluorescence at LAST base
15
Q
next step: polyacrylamide gel
A
- DNA fragments put in gel, migrate based on size
- shorter strands – faster movement
- detector reads color of fluorescent tag as they migrate
- shortest to longest
