molecular techniques

Question 1

what is polymerase chain reaction

Answer 1

it is a technique that amplifies a specific region of DNA from a trace sample

Question 2

outline the steps of polymerase chain reaction

Answer 2

1. heat reaction mixture to 95ºc for 30 seconds to denature DNA sample by breaking hydrogen bonds, obtaining single stranded DNA
2. cool the reaction mixture to 54ºc for 1 minute in the presence of excess primers to allow primers to anneal themselves to complementary sequences at the 3’ OH ends of the DNA strand via formation of hydrogen bonds. Primers flank the sequence of interest.
3. heat reaction mixture to 72ºc for 2 minutes, which is the optimum temperature of TAQ polymerase. The annealed primers prime DNA synthesis using the 4 deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) with TAQ polymerase catalysing the process

Question 3

why is TAQ polymerase used?

Answer 3

TAQ polymerase is stable at high temperatures and hence will not be denatured by repeated heat treatments in the PCR cycle

Question 4

why is PCR called chain reaction

Answer 4

newly synthesised DNA strands serve as template for DNA synthesis in subsequent cycles

Question 5

what are the 2 practical applications of PCR

Answer 5

1. PCR can amplify a small amount of original sample in a short time
2. PCR amplifies specific section of DNA between the 2 primers, hence producing pure PCR products that are exclusive the sequence of interest.

Question 6

what are the 3 advantages of PCR

Answer 6

1. PCR is very sensitive and can amplify sequences from minute amounts of DNA
2. PCR is fast and easy to use
3. PCR is able to amplify sequences from DNA that is degraded, as well as from DNA that is difficult to isolate

Question 7

What are the 5 limitations of PCR

Answer 7

1. It is less effective for longer products due to loss on enzyme activity as well as accumulated inaccuracies
2. TAQ polymerase do not have 3’ to 5’ exonuclease activity and hence error will occur
3. There is a risk of contamination because of the specificity of PCR. Hence, when sample is contaminated by bacteria or viruses, there may be amplification of non-target sequences
4. PCR can only amplify nucleic acids and not proteins.
5. In order to amplify sequences, prior information is needed

Question 8

what is gel electrophoresis

Answer 8

it is the technique of separating charged molecules based on their different rates of movement in an electric field, which is dependent on their size.

Question 9

why will DNA molecules move to the positive electrode?
`

Answer 9

because of the negatively charged phosphate groups on sugar phosphate backbone of DNA`

Question 10

what is the role of the gel matrix in GEL E?

Answer 10

the network of pores in the gel matrix act as a molecular sieve that impedes movement of DNA, separating them by size, because shorter fragments are less impeded by the pores and can move through the gel more quickly, allowing DNA fragments of different lengths to migrate as distinct bands.

Question 11

what are the 10 steps of gel electrophoresis

Answer 11

1. agarose mixture is mixed with buffer solution
2. a gel tray with a gel comb is placed at one end to create wells in the gel and gel solution is allowed to cool and solidify
3. gel tray is filled with electrophoresis buffer solution to cover the gel, allowing electric current to flow through the gel
4. gel comb is removed and wells are available for loading of DNA samples
5. DNA samples are mixed with a loading dye to appear visible and each sample is loaded into a well.
6. DNA ladder is loaded into one of the wells.
7. power is turned on and anode is placed on the opposite end of the gel
8. electrophoresis is allowed to run until DNA ladder travels 2/3 of the length of the gel
9. current is turned off
10. gel is stained with a DNA binding dye such as methylene blue to allow for visualisation of bands

Question 12

what are the 4 uses of GEL E

Answer 12

1. separate DNA fragments according to size
2. determine if a PCR is successful
3. determine the approximate molecular weight of fragments
4. isolate and purify individual DNA fragments

Question 13

what is nucleic acid hybridisation

Answer 13

it is the process by which 2 complementary single stranded nucleic acid chains base pair and reform a double stranded hybrid

Question 13

what is nucleic acid hybridisation

Answer 13

it is the process by which 2 complementary single stranded nucleic acid chains base pair and reform a double stranded hybrid

Question 14

how does nucleic acid hybridisation work

Answer 14

by keeping 2 single stranded nucleic acids at low temperature of 65ºc for a prolonged time, hydrogen bonds between complementary base pairs can re-establish, allowing single strands to re-anneal and reform a double helix

Question 15

what are probes and when are they used

Answer 15

probes are labelled single stranded nucleic acids that are used during hybridisation

Question 16

what are the 2 advantages of nucleic acid hybridisation

Answer 16

1. nucleic acid hybridisation is very sensitive and can detect complementary sequences that are low in concentration
2. nucleic acid hybridisation is selective as the probe can only hybridise a nucleic acid molecule if it carries the complementary sequence

Question 17

what are the 5 uses of nucleic acid hybridisation

Answer 17

1. to detect, characterise and quantify specific nucleotide sequences
2. to locate genes of interest of related but non-identical genes in cells
3. to study gene expression
4. to screen and identify colonies carrying DNA of interest
5. to compare nucleotide sequences in phylogeny studies

Question 18

compare and contrast between probes and primers

Answer 18

• both are short and single stranded nucleotides that bind to DNA via complementary base pairing
• DNA primers bind to a specific sequence on DNA template to provide the 3’ OH group for DNA polymerase to add nucleotides to while a probe allows for detection of target DNA from a sample

Question 19

describe the 8 steps of southern blotting

Answer 19

1. nucleic acid molecules are separated by size using gel e
2. the gel is placed on a paper wick and a sheet of nitrocellulose/ nylon membrane that binds nucleic acid is laid over the gel, followed by blotting paper
3. capillary action draws up the buffer solution through the gel and membrane into the blotting paper.
4. in southern blotting, the alkaline solution denatures the nucleic acids into single strands and the separated DNA molecules are transferred onto the membrane.
5. the nitrocellulose/ nylon membrane is peeled off and incubated with a buffered salt solution containing labelled single stranded probes that has a sequence complementary to the DNA of interest.
6. over a prolonged time, the probe hybridises with the DNA of interest.
7. the membrane is removed and washed to remove unbound DNA
8. autoradiography is carried out so that DNA that has hybridised with the probe shows up as bands