Lab #11

Question 1

Restriction Endonucleases

Answer 1

Enzymes that cut DNA at specific sequences (restriction sites)

–> Cut DNA at internal (endo-) positions

Question 2

What do all recognition sequences for each restriction enzyme have in common?

Answer 2

They are all PALINDROMES

–> Complementary strands are the same (read the same 5’ to 3’)

Question 3

What is a recognition sequence?

Answer 3

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

Question 4

What is a restriction site?

Answer 4

A specific sequence of nucleotides in a DNA molecule that restriction enzymes recognize and cleave

Question 5

What are the different types of cuts that restriction enzymes can make?

What types of fragments do they produce?

Answer 5

1) Blunt cut = Blunt-ends
2) Staggered cut = Sticky-ends: each fragment has an overhang

Question 6

What are Sticky Ends?

Answer 6

Single-stranded overhangs on cut fragments, which are complementary to each other

Question 7

Single-stranded overhangs are referred to by…

Answer 7

The free end of the overhanging strand
–> 5’ overhang or 3’ overhang

Question 8

Which chromosome were we amplifying DNA from?

Answer 8

Chromosome 6

Question 9

How big was the region of DNA we were amplifying

Answer 9

227 base pairs

Question 10

derived Cleaved Amplified Polymorphic Sequence (dCAPS) Assay

Answer 10

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!)

Question 11

Cleaved Amplified Polymorphic Sequence (CAPS)

Answer 11

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!

Question 12

CAPS vs dCAPS

Answer 12

CAPS = detects polymorphisms in already present restriction sites!

dCAPS = detects polymorphisms by INTRODUCING a restriction site (where there wasn’t one previously)

Question 13

SNP

Answer 13

Single-Nucleotide Polymorphism

= A variation in DNA that occurs when a single nucleotide is different from the reference sequence

Question 14

What is different about PCR primers in dCAPs assay?

Answer 14

The primers contain a MISMATCH to the template DNA!

Question 15

Site-directed mutagenesis

Answer 15

A technique that alters the nucleotide sequence of a gene at a specific location

Question 16

What effect does the primer mismatch have on the DNA during PCR?

Answer 16

Causes site-directed mutagenesis
–> Changes the nucleotide sequence during the amplification of the DNA region of interest

Question 17

In our dCAPs assay, which primer had the mismatch? As a result, how did this affect the primer length?

Answer 17

The FORWARD primer had the mismatch

–> Therefore the forward primer was significantly longer than the reverse!

Question 18

If primer has a mismatch, how can it still bind the template strand?

Answer 18

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!

Question 19

What is the recognition site for Hind III? What is the cut made?

Answer 19

A*AGCTT

–> The cut is made between the A’s and therefore forms sticky ends

Question 20

What is the mutation that is inserted by the dCAPs primer?

Answer 20

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

Question 21

If the SNP present in the sample is a G, what will the results of the gel be?

Answer 21

TWO BANDS

(Restriction site AAGCTT was successfully created)

Question 22

If the SNP present in the sample is an A, what will the results of the gel be?

Answer 22

ONE BAND

(Restriction site NOT made as there is no G to create the AAGCTT and instead what is formed is AAACTT)

Question 23

Measuring leaf angle in ImageJ

Answer 23

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

Question 24

Which leaves did we measure the leaf angle for?

Answer 24

The 2nd and 3rd leaves

Question 25

Agarose Gel Electrophoresis

Answer 25

A technique that can separate DNA fragments by size

Question 26

What buffer is the agarose gel immersed in?

Answer 26

1X TAE = Tris-Acetate-EDTA

1X TBE = Tris-Borate-EDTA

Question 27

What is the purpose of loading dye?

Answer 27

Keeps the DNA sample in the gel wells so it does not float out into the buffer by containing a viscous agent (glucose/sucrose)

Question 28

What is added to the samples before loading them into the wells?

Answer 28

Loading Dye

Question 29

What is the voltage charge at each end of the agarose gel?

Answer 29

Well-end = (-) –> Repels the DNA = movement

Opposite-Well-End = (+) –> Attracts the DNA = movement

Question 30

What fluorescent dye was used? WHY?

Answer 30

SYBR Green or SYBR Safe

–> Added so that the bands of DNA fragments could be analyzed using UV light

Question 31

DNA Marker

Answer 31

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

Question 32

How is a 4% agarose gel made?

Answer 32

By adding 2g agarose to 50 mL of 1X TAE buffer and heating/swirling the mixture until clear

Question 33

To what was the SYBR dye added?

Answer 33

Added into the agarose gel before getting set!!!

Question 34

What does the # of bands represent in the gel?

Answer 34

The number of DNA fragments!

Question 35

Primers are always WRITTEN:

Answer 35

5’ to 3’

Question 36

What is an amplicon?

Answer 36

The end product of PCR –> an amplified piece of DNA

Question 37

How do you calculate length of amplicon?

Answer 37

(HIGHEST end of REVERSE primer) - (LOWEST end of FORWARD primer) + 1

Question 38

If no restriction site is created, the sample has the ______ SNP and produces _______ band/s in the gel with size/s__________

Answer 38

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

Question 39

If a restriction site IS created, the sample has the ______ SNP and produces _______ band/s in the gel with size/s__________

Answer 39

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

Question 40

When running a DNA gel, why should the percentage of an agarose be changed?

Answer 40

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