Ignore - Optional Tutorial 2: Fundamental Molecular Techniques

Question 1

Describe PCR

Answer 1

Polymerase chain reaction = uses the natural mechanisms of DNA replication combined with hybridization to replicate a certain DNA sequence

Question 2

What are the 5 reagents/materials needed for PCR?

Answer 2

1. Sample DNA = template DNA
2. DNA primers that give the DNA pol a foothold to actually replicate the DNA
3. Deoxy-nucleoside triphosphates (dNTP) = the 4 types of nucleotides that act as the building blocks for DNA
4. DNA polymerase = the enzyme that takes the dNTPs and puts them together to produce the complementary DNA strand
5. Buffer solution that is used to regulate the temp for optimal DNA polymerase activity

Question 3

What are the steps to PCR? include the temps

Answer 3

1. Denaturation = splitting the dsDNA to 2 ssDNA by INC the temp (95 degrees)
2. annealing = sticking a complementary DNA primer to the sample DNA (50 - 60 degrees)
3. elongation = the DNA polymerase uses the sample DNA as a template to add the corresponding dNTPs (72 degrees)

Question 4

What is the prupsoe of thermocycler?

Answer 4

used to regulate/set the temp at certain times a certain amount of times during PCR

Question 5

PCR exponentially amplifies sample DNA. What does this mean/how does this happen?

Answer 5

this means that the continuous use of PCR allows for the exponential growth/amplification of a certain sample of DNA. This happens because after the first PCR occurs the number of sample DNAs double, and those 2 sample DNA are then denatured and used as template DNA to then produce 4 sample DNA. This continues to occur resulting in exponential growth (2^n)

Question 6

How many copies of DNA would be produced after 10 cycles of PCR?

Answer 6

the exponential growth doubles every time therefore the formula would be 2^n, where n is the number of PCR cycles
= 2^n
= 2^10
= 1024 copies

Question 7

How many DNA copies would be produced after 35 cycles of PCR?

Answer 7

the exponential growth doubles every time therefore the formula would be 2^n, where n is the number of PCR cycles
= 2^n
= 2^35
= 3.4 x10^10

Question 8

T or F - for the PCR to occur a DNA primer is required per rxn

Answer 8

F - the PCR requires two primers a forward and a reverse

Question 9

Describe the difference b/w a forward and reverse primer including;
a) which strand it anneals to (top/bottom)
b) it has the exact sequence of which strand (top/bottom)

Answer 9

forward
a) bottom
b) top
reverse
a) top
b) bottom

Question 10

what is considered the flanking region?

Answer 10

the sequence of DNA that overhangs past the target sequence and primers.

Question 11

T or F - all PCR cycles produce target copies

Answer 11

F - only the 3rd cycle will. The first 2 will include flank DNA

Question 12

T or F - when companies produce primers to be used to help PCR target a specific DNA sequence there can be some instances when the forward and reverse primers anneal to each other due to being complimentary

Answer 12

T - This can occur however companies try to make sure this doesn’t happen as it will create errors during replication

Question 13

What is DNA sequencing?

Answer 13

determining the sequence of a fragment/entire genome of DNA

Question 14

What are the steps to Sangers sequencing tech?

Answer 14

1. take four rxn that contain high [template], primers, + DNA polymerase (produce complementary strand)
2. add low [ddNTP] in each rxn putting different nucleotides in each (ddNTP are dNTP that or deoxygenated at 3’ end so no most can be added)
3. take the fragments of all 4 complementary strands and place them in gel electrophoresis to separate via size (column for each type of ddNTP)

Question 15

How do you determine the first 3 dNTP of the sequence when using the sanger method?

Answer 15

the column ddNTP that travels the most (smallest) in the gel electrophoresis is the first dNTP in the sequence. The column with the band that traveled the second furthest (second smallest) would be the second dNTP in the sequence. Lastly, the column with the band that traveled the third furthest (third smallest) would be the third. This would continue on for the whole fragment to determine the rest.

Question 16

T or F - the sequence that is read using the sanger method matches the template strand

Answer 16

F - the sequence matches the complementary strand

Question 17

What are the 3 steps to finding the template sequence using the Sanger method?

Answer 17

1. complementary strand (5’ to 3’) = sequence using smallest fragment to biggest (bottom to top)
2. template strand in reverse (3’ to 5’) = matches the bases of the complementary strand
3. template strand (5’ to 3’) = reverse the previews sequence to find the template sequence

PS - step 2 and 3 can be switched and produce the same result

Question 18

What is the main difference b/w a chromatogram and an autoradiogram? How does reading them differ?

Answer 18

chromatogram = displays ddNTPs in fluorescent peak due to being fluorescently labeled. They are read left to right using the corresponding fluorescent colours for each ddNTP type

autoradiogram = displays the ddNTP as bands in the gel due to being radiolabeled. They are read from bottom to top (smallest fragment to largest

Question 19

Describe the 6 steps to the ‘Next generation sequencing”

Answer 19

1. Fragmentation of DNA
2. anneal adapter that contains primer sequences to fragment
3. bind fragment to DNA capture bead (binds to primer sequence end) and place in a sol’n that contains PCR (polymerase chain rxn)
4. PCR amplifies the DNA fragment
5. DNA is placed in a well that contains sequencing reagents
6. DNA synthesis w/in each well

Question 20

In the 5th step of ‘Next generation sequencing,’ it states that the DNA bead that contains the single DNA strands is placed in a well that contains sequencing reagents. What does this mean?

Answer 20

The well will contain reagents such as dNPTs, DNA ligase, DNA polymerase, DNA primase etc which are needed for DNA synthesis

Question 21

Describe emulsion PCR.

Answer 21

a technique used to amplify a fragment of DNA
(ASK PROF)

Question 22

How are the Sanger sequencing and the next-generation techniques similar?

Answer 22

they both utilize sequencing by synthesis using sequencing reagents such as nucleotides, DNA polymerase, and sample DNA.

Question 23

What are 2 next-generation techniques? Describe

Answer 23

1. pyrosequencing = the sequence is washed over by one of the four dNTPs and checked to see if it is the proper base pair (binding result in the flash of light). If not the dNTP used will degrade and another dNTP is washed over the fragment. This will repeat until a flash is present (rxn when the diphosphate is released from the dNTP) then continue for the next nucleotide in the fragment.
2. Reversible Terminator Sequencing = the fragment is washed by all four nucleotides that are fluorescently labeled then a blocking group is added to temporarily prevent any more nucleotides from being added. The colour is recorded and then the blocker is removed.

Question 24

Both the Sanger sequencing method as well as the Next generation DNA Sequencing methods are used to sequence a specific DNA. Each have 2 types associated with how they sythesis the sequence. Name and describe all 4 types, include which method they are associated with.

Answer 24

1. sanger method using gel electrophoresis technique = this involves placing [low] of mutated dNTPs of each type in 4 separate rxns that contain the DNA fragment. The ddNTP (mutated dNTP) will bind to whatever base it pairs with while the DNA polymerase synthesizes it and prevent further elongation. Then the 4 rxns are transferred to a gel electrophoresis where it is displayed by an autoradiogram. Read bottom to top (smallest to largest)
2. Sanger method using chromatogram which fluorescently labels the ddNTP mixed with one rxn of DNA fragment. Followed by the chromatogram reading of the fluorescence peaks that show the end of each fragment. Read left to right based on the colour of specific ddNTP (each dNTP have different fluorescent colours)
3. Next generation technique using pyrosequencing where an unaltered dNTP is washed with the DNA fragment and will show a flash of light (due to release of diphosphate gr) if the proper base pair. If not it will degrade and another dNTP will be washed over the DNA fragment. This will continue until the who sequence is completed. Read left to right based on fluorescent colour (each base has a differ colour)
4. Reversible terminator sequence = where all 4 dNTPs are washed over the DNA fragment (each fluorescently labeled by a differ colour). After a dNTP pair with one of the bases in the sequence, a blocker is temporarily added to stop the addition of more bases until the fluorescent light is read. Then the blocker is removed and the cycle continues.