Lab #11 Flashcards
Restriction Endonucleases
Enzymes that cut DNA at specific sequences (restriction sites)
–> Cut DNA at internal (endo-) positions
What do all recognition sequences for each restriction enzyme have in common?
They are all PALINDROMES
–> Complementary strands are the same (read the same 5’ to 3’)
What is a recognition sequence?
A short DNA sequence that is recognized by a restriction enzyme or DNA-binding protein
–> Once recognized, it leads to the binding of the enzyme/protein to the DNA
What is a restriction site?
A specific sequence of nucleotides in a DNA molecule that restriction enzymes recognize and cleave
What are the different types of cuts that restriction enzymes can make?
What types of fragments do they produce?
1) Blunt cut = Blunt-ends
2) Staggered cut = Sticky-ends: each fragment has an overhang
What are Sticky Ends?
Single-stranded overhangs on cut fragments, which are complementary to each other
Single-stranded overhangs are referred to by…
The free end of the overhanging strand
–> 5’ overhang or 3’ overhang
Which chromosome were we amplifying DNA from?
Chromosome 6
How big was the region of DNA we were amplifying
227 base pairs
derived Cleaved Amplified Polymorphic Sequence (dCAPS) Assay
A slightly altered CAPS technique to also detect SNPs!
POTENTIALLY introduces a restriction site at an SNP site using specific primers that contain mismatches to the template DNA
–> Then analyzes the fragmentation of DNA to determine sequence (by observing whether or not the DNA was cut and therefore whether or not the restriction site was formed!)
Cleaved Amplified Polymorphic Sequence (CAPS)
A technique to detect SNPs!
–> Introduces DNA segment to restriction enzyme and then analyzes if fragmentation occurs or not
–> Gene sequence can therefore be determined based upon whether cutting occurs or not!
CAPS vs dCAPS
CAPS = detects polymorphisms in already present restriction sites!
dCAPS = detects polymorphisms by INTRODUCING a restriction site (where there wasn’t one previously)
SNP
Single-Nucleotide Polymorphism
= A variation in DNA that occurs when a single nucleotide is different from the reference sequence
What is different about PCR primers in dCAPs assay?
The primers contain a MISMATCH to the template DNA!
Site-directed mutagenesis
A technique that alters the nucleotide sequence of a gene at a specific location
What effect does the primer mismatch have on the DNA during PCR?
Causes site-directed mutagenesis
–> Changes the nucleotide sequence during the amplification of the DNA region of interest
In our dCAPs assay, which primer had the mismatch? As a result, how did this affect the primer length?
The FORWARD primer had the mismatch
–> Therefore the forward primer was significantly longer than the reverse!
If primer has a mismatch, how can it still bind the template strand?
Primers don’t always have to match exactly, except the most 3’ prime nucleotide
–> the strength of binding elsewhere on the primer needs to compensate for the instability of the mismatch
–> SOOO having more “matched” bases on the primer = > SA that is properly matched = outweighs the mismatch instability and allows the primer to bind!
What is the recognition site for Hind III? What is the cut made?
A*AGCTT
–> The cut is made between the A’s and therefore forms sticky ends
What is the mutation that is inserted by the dCAPs primer?
The addition of an A!
Original Sequences could have been (following the primer substituted A –> (AAGCTT : the second A in the recognition sequence was the the substituted one)
1) G
2) A
–> Depending on which SNP is present, the restriction site will either be successfully created or not
If the SNP present in the sample is a G, what will the results of the gel be?
TWO BANDS
(Restriction site AAGCTT was successfully created)
If the SNP present in the sample is an A, what will the results of the gel be?
ONE BAND
(Restriction site NOT made as there is no G to create the AAGCTT and instead what is formed is AAACTT)
Measuring leaf angle in ImageJ
1) Select angle symbol
2) Find the leaf blade (2nd or 3rd) and position the cursor PARALLEL to the midrib before the leaf begins to curve (plot a point.
3) drag this down to a second plotted point at the middle of the ligule region
4) Hold down the shift key and create a line going straight up from that ligule point
5) Hit the measure button and receive the value
Which leaves did we measure the leaf angle for?
The 2nd and 3rd leaves
Agarose Gel Electrophoresis
A technique that can separate DNA fragments by size
What buffer is the agarose gel immersed in?
1X TAE = Tris-Acetate-EDTA
1X TBE = Tris-Borate-EDTA
What is the purpose of loading dye?
Keeps the DNA sample in the gel wells so it does not float out into the buffer by containing a viscous agent (glucose/sucrose)
What is added to the samples before loading them into the wells?
Loading Dye
What is the voltage charge at each end of the agarose gel?
Well-end = (-) –> Repels the DNA = movement
Opposite-Well-End = (+) –> Attracts the DNA = movement
What fluorescent dye was used? WHY?
SYBR Green or SYBR Safe
–> Added so that the bands of DNA fragments could be analyzed using UV light
DNA Marker
A sample made up of a mixture of DNA fragments of known sizes that is run in one of the lanes of the gel
–>The size of each unknown DNA fragment from your PCR reactions can be estimated by comparing it to the known sizes of the fragments in the DNA marker run in an adjacent lane of the gel
How is a 4% agarose gel made?
By adding 2g agarose to 50 mL of 1X TAE buffer and heating/swirling the mixture until clear
To what was the SYBR dye added?
Added into the agarose gel before getting set!!!
What does the # of bands represent in the gel?
The number of DNA fragments!
Primers are always WRITTEN:
5’ to 3’
What is an amplicon?
The end product of PCR –> an amplified piece of DNA
How do you calculate length of amplicon?
(HIGHEST end of REVERSE primer) - (LOWEST end of FORWARD primer) + 1
If no restriction site is created, the sample has the ______ SNP and produces _______ band/s in the gel with size/s__________
The sample has the A SNP (AAACTT = no restriction site) and therefore produces ONE band in the gel for ONE fragment that is 227 BP in length
If a restriction site IS created, the sample has the ______ SNP and produces _______ band/s in the gel with size/s__________
The sample has the G SNP (AAGCTT = restriction site) and therefore produces TWO bands in the gel for TWO fragment that have the lengths: 43 + 184 BP
When running a DNA gel, why should the percentage of an agarose be changed?
The percentage of agarose in a DNA gel should be changed depending on the size of the DNA fragments you want to separate, as a higher agarose concentration creates smaller pores in the gel, allowing for better resolution of smaller DNA fragments, while a lower concentration is better for separating larger DNA fragments
