Lecture 11-MT2 Flashcards
What direction does the primer strand go in?
5’ to 3’
What direction does the template strand go in?
3’ to 5’
Which strand is complementary and has free 3’ OH?
primer strand
What do we need for DNA replication?
-DNA
-DNA polymerase
-Enzymes to help DNA polymerase
-Primers
-dNTPs
What does PCR need?
-DNA
-dNTPs
-DNA pol
-DNA primers (short, around 20 bp)
What are PCR steps
- Melting/denaturation (95 degrees celsius)
- Annealing (around 60 degrees celsius)
- Elongation (72 degrees celsius)
- Repeat 20-30 times
Why is denaturation at 95 degrees celsius?
This temp breaks down hydrogen bonds and separates 2 strands
For the annealing step why does temperature vary and depend on bases?
Temperature for annealing depends on bases b/c
-C-G base pairing has more hydrogen bonds (3)
-A-T has less (2) bonds
-it depends what is in DNA, more heat to break down more bonds
Why is elongation always at 72 degrees celsius?
Optimal temp for TAQ polymerase (polymerase used in PCR)
What is the order of PCR?
Denaturation, annealing, elongation
What is the average size of CRISPR locus?
800-2000 bp
Where do primers bind in CRISPR locus
100 bp upstream and downstream
How are DNA samples separated?
By size
What is electrophoresis?
Separates DNA molecules on an electric field
In electrophoresis chamber we load the DNA on…
The (-) pole since DNA is (-) it will be attracted to the (+) side & migrate through the gel & be separated by size
True or false: large fragments move faster through gel
False
-small fragments go faster
What does a bigger fragment size in gel indicate?
The bigger the size the more spacers it has
What is used for Sanger sequencing?
-1 primer
-makes ssDNA pieces
-not exponential
-2 kinds of nucleotides (regular and ddNTPs)
What are dideoxynucleotides? (ddNTPs)
Used in Sanger sequencing
- does not have 3’ OH so no more nucleotides can be added–>termination of primer chain extension
Why do we amplify DNA sequences?
Eliminates noise so we can have correct fragments
What is Sanger’s method?
-modified PCR
-Electrophoresis
-Sequence determination
How is electrophoresis modified in Sanger’s method?
Electrophoresis uses a modified laser beam
-when fragment reaches beam a fluorescent color corresponds to base
True or false: Sanger sequencing uses large fragments, such as over 1000 bp
False
-Sanger sequencing uses small regions of DNA (800-1000 bp)
-if larger than 1000 you can miss a part of sequence
Do you expect the sizes of the CRISPR loci analyzed
through electrophoresis (bands) to correspond to the sizes
of the CRISPR loci’s sequences (the ones sequenced)
No
-If sequences are too large they will not appear
