Molecular Techniques

Question 1

what is the purpose of the polymerase chain reaction (PCR)?

Answer 1

it can amplify a specific region of DNA from a trace sample

Question 2

what are the conditions required for amplification to occur?

Answer 2

it must be carried out in vitro (outside the living organism), with heat used to separate the 2 DNA strands by breaking hydrogen bonds
at least part of the sequence of the DNA sample must be known

Question 3

what are the five components of polymerase chain reaction?

Answer 3

DNA template: double-stranded DNA sample containing the nucleotide sequence of interest
PCR primers: two sets of short, single-stranded DNA specific to the sequence and flanking it, complementary to the 3’ end of both template strands
free deoxyribonucleoside triphosphates / dNTPs (dATP, dTTP, dCTP, dGTP): present in excess for synthesis as raw materials for synthesis of new DNA strands
thermostable DNA polymerase: thermally-stable, not denatured by repeated heat treatments (eg. Taq polymerase)
PCR reaction buffer: made of buffering salts and detergent, and Mg2+ ions as cofactors for DNA polymerase activity.

Question 4

state the three steps in a single cycle of the polymerase chain reaction

Answer 4

1. denaturing of DNA template
2. annealing of primers
3. extension of primers

Question 5

what occurs during the first step of a cycle of PCR?

Answer 5

denaturation of DNA template-
occurs between 90-100 degrees Celcius
heated to 95 degrees C for 30s
hydrogen bonds holding two strands of DNA template broken, denaturing it and forming single-stranded DNA

Question 6

what occurs during the second step of a cycle of PCR?

Answer 6

annealing of primers-
occurs between 50-65 degrees Celcius
cooled to 54 degrees C for 1 min in presence of large excess of both DNA primers
cooling allows primers to anneal specifically to complementary sequences at 3’ end of single-stranded DNA templates via hydrogen bonding (hybridisation)

Question 7

what occurs during the third step of a cycle of PCR?

Answer 7

extension of primers-
occurs between 60-75 degrees Celcius
heated to around 72 degrees C for 2 min
close to optimum temperature of thermostable Taq polymerase
annealed primers prime DNA synthesis using four deoxyribonucleoside triphosphates, catalysed by Taq polymerase
region of DNA downstream of each primer extended in 5’ to 3’ direction

Question 8

during polymerase chain reaction, what temperature changes occur to each mixture, and how are they achieved?

Answer 8

denaturation of DNA template: heated to 95 degrees C for 30s
annealing of primers: cooled to 54 degrees C for 1 min
extension of primers: heated to around 72 degrees C for 2 min

Question 9

what are the four features of polymerase chain reaction?

Answer 9

1. chain reaction: newly synthesised DNA strands serve as templates for DNA synthesis in subsequent cycles
2. PCR is specific: only gene of interest amplified
3. 30 cycles: almost all DNA molecules are exact copies of target sequence then
4. number of DNA molecules after 30 cycles has increased exponentially to over 1 billion

Question 10

what are the practical applications of PCR?

Answer 10

PCR can specifically amplify DNA sequences in a short time, making large amounts of pure PCR products (almost exclusively the sequence of interest)
forensic analysis, medical testing, detection of infectious diseases

Question 11

what are the three advantages of the polymerase chain reaction

Answer 11

sensitivity: can amplify sequences from minute amounts of DNA
speed and ease of test: rapid, easily automated (30 cycles around 3 hours)
robustness: amplification from DNA that is badly degraded or embedded in medium from which conventional DNA isolation is difficult, suitable for paleontology, anthropology, pathology studies

Question 12

what are 5 limitations of the polymerase chain reaction?

Answer 12

1. risk of contamination: non-target sequences from non-template nucleic acids can be accidentally amplified instead
2. infidelity of DNA replication in vitro: DNA polymerases lack 3’ to 5; exonuclease activity (proofreading)
3. short size and limiting amounts of PCR product: efficient amplification only for products up to a few thousand base-pairs
4. need for target DNA sequence information: to construct specific oligonucleotide primers
5. only for nucleic acids (DNA / RNA), not proteins

Question 13

how does gel electrophoresis work?

Answer 13

it separates molecules based on their different rates of movement / migration in an electric field due to charge or size
by applying direct current through semi-solid, porous gel matrix made of agarose and polyacrylamide to make a complex network of pores that act as a “molecular sieve”)

Question 14

describe how linear DNA molecules are separated in gel electrophoresis (direction of movement, basis of separation by molecular sieve)

Answer 14

based on their rate of movement through the gel matrix, (not charge bc they essentially have constant charge density), depending on their size
shorter DNA fragments less impeded by pores than longer ones, move through gel more quickly, size-fractionating complex mixture of linear DNA fragments into discrete bands
since phosphate groups of DNA’s sugar-phosphate backbone are negatively-charged, DNA moves towards positive electrode (anode)

Question 15

what makes up the molecular sieve in gel electrophoresis?

Answer 15

agarose: polysaccharide extracted from seaweed (more porous, separates larger DNA molecules)
polyacrylamide: from monomers of a small organic molecule, acrylamide (less porous, separates smaller DNA molecules)

Question 16

what happens to the gel after electrophoresis, to visualise the separated DNA fragments?

Answer 16

stained with DNA-binding dye
- methylene blue
- ethidium bromide (intercalates DNA and fluoresces in UV light)

Question 17

what are the four practical applications of DNA gel electrophoresis?

Answer 17

1. to separate DNA fragments according to size
2. to determine approximate molecular weight of separated DNA fragments
3. to isolate / purify individual DNA fragments
4. to check fi PCR experiment was successful

Question 18

state the five steps of gel electrophoresis

Answer 18

1. preparing gel with wells for DNA samples
2. setting up of gel for electrophoresis
3. loading DNA samples into wells
4. application of electric field to start electrophoresis
5. staining of gel to view separated DNA bands

Question 19

what are the steps to prepare the gel for electrophoresis (before loading DNA samples)?

Answer 19

mix agarose powder with a buffer solution (to stabilise DNA during electrophoresis), heat till agarose dissolves
gel tray with gel comb to create wells in gel, pour the solution into the tray and allow to cool and solidify

after gel has solidified, place tray with gel into an electrophoresis chamber with enough electrophoresis buffer solution to cover the gel, allows electric current to flow through the gel from electrodes at one end to the other
remove comb to leave wells for loading of DNA samples

Question 20

why are DNA samples mixed with a loading dye, and what is the dye made of?

Answer 20

DNA samples mixed with loading dye (bromophenol blue + xylene cyanol + glycerol, different from DNA-binding dye later), makes DNA sample visible
bromophenol blue moves slightly faster than DNA, xylene cyanol moves slightly slower, helps monitor rate of DNA movement
glycerol increases density of DNA sample, so they sink to bottom of well when loaded

Question 21

how is an electric field applied to start electrophoresis?

Answer 21

DC power supply connected to electrodes, positive anode opposite DNA samples, moves from cathode to anode (negatively-charged sugar-phosphate backbones)
conducted till DNA molecules of different sizes are well separated, dye marker has moved a suitable distance (about 2/3 length of gel), current is turned off and gel removed.

Question 22

what is a DNA ladder, and must each gel run be done with one in gel electrophoresis?

Answer 22

DNA ladder / molecular weight marker is a known DNA molecule that has been digested into fragments of known size by a known restriction enzyme (eg. lambda phage DNA, digested by Hind III).
to estimate size of DNA fragments in the sample lanes

Question 23

define nucleic acid hybridisation

Answer 23

nucleic acid hybridisation is the process by which two complementary, single-stranded nucleic acid chains base-pair and reform a double-stranded hybrid

Question 24

what two processes does nucleic acid hybridisation include, and what are the conditions for them to occur?

Answer 24

DNA denaturation / melting: DNA double helix can be separated into two single strands under conditions that disrupt the hydrogen bonds (eg. heating to around 100 degrees Celsius, high pH of >13, or very low salt concentrations)
DNA renaturation / hybridisation: when kept for a prolonged period at lower temperature of 65 degrees Celsius, hydrogen bonds between complementary base-pairs can re-establish to reanneal, reforming a double helix

Question 25

what are the applications of nucleic acid hybridisation, and what makes the reaction more favourable?

Answer 25

• detect, characterise, quantify specific DNA and RNA base / nucleotide sequences using specific single-stranded nucleic acid probes of known sequence
• locate particular genes of interest
  to study gene expression and changes in gene expression profiles
• screen libraries of cloned DNA or bacterial clones to identify clones / colonies
• compare base nucleotide sequences

the greater the complementarity between both single-stranded molecules, the more favorable the hybridisation reaction

Question 26

what are probe molecules in nucleic acid hybridisation?

Answer 26

single-stranded DNA or RNA
from 15 to several thousand nucleotides long
labelled (with radioactive / fluorescent / chemical markers to facilitate detection)
can be cloned from genomic or cDNA molecules, fragments generated by PCR, or chemically synthesised nucleic acid molecules

Question 27

what are the advantages of nucleic acid hybridisation?

Answer 27

sensitive: complementary sequences present at a conc as low as one molecule per cell can be detected
selective: probe hybridises only to nucleic acid molecules carrying all or part of the complementary sequence
do not need to be perfectly complementary, stringency of hybridisation can be controlled by varying incubation temperatures

Question 28

what are the similarities and differences of the structures and functions between a primer (used in PCR) and a probe (used in nucleic acid hybridisation)?

Answer 28

similarities: short, single-stranded oligonucleotides that bind to DNA via complementary BP
differences: primer provides free 3’-OH group for DNA replication, probe allows detection fo target DNA from sample
probe also structurally modified (labelled), primer is not

Question 29

what are the four practical steps of southern blotting?

Answer 29

1. agarose gel electrophoresis: nucleic acids (dsDNA molecules) separated on basis of size
2. blotting:
3. incubation with labelled probe
4. detection of bound probe

Question 30

how does blotting occur during southern blotting?

Answer 30

• gel after gel e is placed on a paper wick
• absorbs buffer solution from the reservoir
• with sheet of either nitrocellulose or nylon membrane which binds nucleic acid and stack of blotting paper
• capillary action draws the buffer solution through the gel and nitrocellulose / nylon into the blotting paper
• alkaline condition of buffer denatures the dsDNA into single-stranded DNA
• separated DNA molecules are blotted onto nitrocellulose / nylon membrane