3.5 Molecular biology techniques

Question 1

What are the 3 types of molecular techniques?

Answer 1

1. Polymerase chain reaction (PCR)
2. Gel Electrophoresis
3. Southern blotting

Question 2

What is PCR?

Answer 2

PCR is a technique that allows for the amplification of a specified segment of DNA in vitro

Question 3

What are the 5 components of the reaction mixture for PCR?

Answer 3

1. Template DNA
   DNA containing the segment to be amplified; usually genomic DNA, plasmid or fragmented DNA
2. Oligonucleotide primers
   Synthetic single-stranded DNA primers are typically 20-30 nucleotides long and are needed to initiate DNA synthesis
   Primers have sequences complementary to sequences at 3’ ends of both strands of target DNA sequence
   Two different primers are needed, each one complementary and able to bind to the 3’ ends of each template strand of the DNA double helix
   Knowledge of sequences flanking the DNA segment of interest is crucial to artificially synthesise the primers
   Primers are present in large excess to increase the likelihood of primers binding to target DNA (relative to template strands reannealing to each other again)
   Primers become part of the amplified segments
3. Taq polymerase
   Thermostable DNA polymerase
   Stable at 95 and works optimally at 72

4. DeoxyribonucleoSide triphosphates (dNTPs)
dATP
dTTP
dCTP
dGTP
substrates for DNA replication

5. Buffer containing Mg2+
   cofactor for proper DNA polymerase function

Question 4

PCR protocol

Answer 4

1. Place all components into a PCR tube and place the tube into the thermocycler and run a standard programme that involves heating the tube to different temperatures for different periods of time
2. Cycle:
   A) Denaturation:
   Brief heat treatment up to 95 to denature and separate the two strands of DNA double helix, exposing nitrogenous bases for complementary base pairing required in annealing and extension
   B) Primer annealing:
   cooling of DNA e.g. 64 in the presence of a large excess of DNA primers, allowing primers to reanneal to complementary sequences at 3’ ends. of each template DNA strand
   C) Extension:
   Taq polymerase synthesises complementary DNA strand by catalysing formation of phosphodiester bonds between dNTPs at an optimum of 72
   Chain extension occurs from 3’ end of DNA primer which provides free 3’ OH group required by Taq polymerase

Question 5

What is the result of PCR?

Answer 5

Each cycle results in a doubling in the number of DNA molecules being replicated
n cycles = 2^n molecules of target DNA
Amount of desired sequence increases exponentially

Question 6

Why is it usually recommended to run about. 25-30 cycles for PCR?

Answer 6

Fewer cycles: insufficient DNA for analysis

Too many cycles: Too many mistakes in replication

Question 7

What are the advantages of PCR?

Answer 7

1. Sensitivity:
   only a minute amount of source DNA is required (number of copies of target DNA doubles with each cycle; exponential increase even from low copies of starting material)
2. Speed:
   only a few hours are required to generate enough amplified DNA compared to multiple steps of cloning which need at least a week

Question 8

What are the applications of PCR?

Answer 8

1. Clinical diagnosis (pre-natal screening and early detection of infection)
2. Forensics (Identification of suspects)
3. Evolution (Study evolutionary relatedness)

Question 9

What are the limitations of PCR?

Answer 9

1. Taq polymerase lacks 3’ to 5’ proofreading ability
   Taq polymerase cannot check or remove an incorrectly incorporated nucleotide
   Errors occuring early in PCR reaction will get compounded with each replication cycle as all daughter molecules resulting from this early error will be exponentially affected
2. Synthesis of PCR primers depends on sequence information from target region
   Requires knowledge of sequences flanking target region
   If primers are designed incorrectly/ contain non-specific sequences, then no amplification occurs/ wrong DNA fragments may be amplified
3. Limit to size of DNA fragment to be amplified:
   DNA fragments to be amplified are limited to about 3kb
   Further increase in length of target sequence decreases efficiency of amplification
   Polymerase tends to fall off DNA template before chain extension is complete
4. Exponential amplification of contaminant DNA:
   It is possible to contaminate a fresh PCR reaction with minute amounts of contaminant DNA due to poor lab skills
   such unwanted DNA sequences may be amplified to significant amounts, alongside target DNA sequences

Question 10

What is Gel electrophoresis?

Answer 10

Gel electrophoresis is a technique that separates mixtures of DNA, RNA or proteins according to their molecular size

Question 11

What is the principle of Gel electrophoresis?

Answer 11

Electrophoresis is movement of charged molecules in an electric field
Biological molecules such as DNA, RNA and proteins exist in solution as electrically-charged particles at a given pH

Question 12

How do DNA molecules travel in gel electrophoresis?

Answer 12

When placed in an electric field, negatively-charged DNA molecules will move away from the negative electrode (cathode) and move towards the positive electrode (anode)

Question 13

What forms the gel and how does it allow electrophoresis to function?

Answer 13

Agarose
Meshwork of polymer fibers that makes up agarose gel impedes movement of DNA fragments, impeding longer fragments more than shorter ones allowing DNA fragments of different sizes to be separated out based on their different rates of migration

Question 14

What is the protocol for gel electrophoresis?

Answer 14

1. A slab of agarose gel is placed in a buffer solution which allows conduction of electricity to generate an electric field. The gel has been pre-cast with little indentations called wells at one end of the gel slab using a comb. DNA samples are loaded into these wells
2. Prior to loading, DNA sample is first mixed with a dense loading dye which helps DNA sample sink to the bottom of the well. Loading dye contains 2 different coloured loading dyes which also travel along in the gel just like the DNA sample does during electrophoresis
3. As DNA is invisible to the naked eye, the 2 loading dyes help to monitor the progress of electrophoresis; usually, dark blue dye corresponds to 100bp while light blue dye corresponds to 1100bp
4. Usually a DNA ladder is loaded into the gel; this is a mixture of DNA fragments of known sizes which act as a standard that can be compared with fragment of unknown sizes in the sample
5. When the current is turned on, all DNA fragments migrate out of the well into the agarose. DNA is attracted towards the positive electrode due to its negative charge
6. Meshwork of polymer fibers that make up agarose gel impedes movement of longer DNA fragments more than shorter ones and thus shorter fragments will migrate towards the positive electrode at a higher ratee
7. The fragments of different sizes in the DNA sample separate out in the gel based on their different rates of migration. If the amount of fragments is high enough, these are seen as discrete bands on the gel after staining with each band consisting of DNA molecules of the same length
8. before loading dye reaches the end of the gel, current is switched off
9. To visualise the bands, the gel slab is stained with DNA-binding dye (usually ethidium bromide) and placed under UV light under which the DNA bands will be revealed as the dye bound to DNA fluoresces
10. It is possible to alter the resolution of the gel by varying the concentration of agarose used. The higher the concentration, the better the resolution i.e. even fragments of very small differences in size can be separated

Question 15

What are the uses of electrophoresis?

Answer 15

To estimate…
1. Fragment size
2. Amount of DNA
To analysee and very. DNA fragments such as PCR products and restriction fragments

Question 16

What is Southern Blotting?

Answer 16

Typically carried out after gel electrophoresis, it is a technique used to detect and confirm fragments containing specific nucleotide sequences within a sample of DNA

Question 17

What is the protocol for southern blotting?

Answer 17

1. After DNA samplee is amplified by PCR or cut by restriction enzymes, DNA fragments are separated by electrophoresis
2. The gel slab containing DNA fragments is placed on top of an absorbent sponge in a tray of alkaline solution. A nitrocellulose membrane and a stack of paper towels are placed on top of the gel slab. A heavy weight is stacked on top of the paper towels
   [heavy weight > paper towels > nitrocellulose membrane > gel slab > sponge in alkaline solution]
3. The absorbent paper towels draw the alkaline solution towards themselves. This. capillary action draws alkaline solution upwards through the gel; Alkaline solution denatures the double-stranded DNA fragments in the gel into single-stranded DNA which are then drawn upwards onto the nitrocellulose membrane and binds to it (in the same position as in the gel)
4. Nitrocellulose membrane is removed and incubated with a radioactive single-stranded DNA probe which is complementary in sequence to part of the target sequence. DNA fragments containing this part of the target sequence hybridise to the probe by complementary base pairing
5. After hybridisation, the membrane is washed to remove any unhybridised probes
6. Autoradiography is performed by placing an X-ray film over the membrane; radioactivity of bound probes exposes the film forming an image corresponding to the bands that have hybridised to the probe

Question 18

Where are restriction enzymes derived?

Answer 18

Bacteria

Question 19

What is the action of restriction enzymes?

Answer 19

Recognise and bind to specific nucleotide sequences known as restriction sites in double stranded DNA and cleave phosphodiester bonds on both strands of the DNA
Some restriction enzymes produce sticky ends while some produce blunt ends

Question 20

Why is the bacterium’s own DNA not cleaved?

Answer 20

It is methylated at restriction sites recognised by its own restriction enzymes
An enzyme adds methyl groups (-CH3) to adenine or cytosine residues in DNA sequence that constitutes the restriction site. This changes the restriction site such that it is no longer complementary in conformation and charge to the restriction enzyme’s active site. Hence, the restriction enzyme would be unable to recognise and cleave the DNA

Question 21

What is DNA polymorphism?

Answer 21

DNA polymorphism refers to differences in DNA sequences in homologous regions among different individuals; either in nucleotide sequence or in number of tandemly repeated nucleotide units

Question 22

What are the possible differences and what are the results of such differences? [DNA polymorphism]

Answer 22

1. Single nucleotide polymorphism (SNPs)
   Difference in a single base-pair in homologous regions of different indivuals
   Results in variation in number and location of restriction sites
   Produces fragments of different lengths from different individuals
2. Presence of variable number tandem repeats (VNTRs) in homologous regions of different individuals
   Results in variation in number and location of restriction sites
   Produces fragments of different lengths from different individuals

Question 23

What is RFLP?

Answer 23

Restriction fragment length polymorphism:
Unique banding pattern among individuals when their genomic DNA is digested by restriction enzymes, separated by gel electrophoresis and subjected to Southern hybridisation

Question 24

Applications of RFLP

Answer 24

1. Disease detection e.g. sickle cell anaemia: detecting SNPs
2. DNA fingerprinting: detecting VNTRs

Question 25

Describe the method(s) for detecting SNPs

Answer 25

Method 1:
Genomic DNA from an individual with sickle cell anemia and a normal individual are isolated
1. Digest with restriciton enzyme, Mstll
Different sized restriction fragments are produced
2. Gel electrophoresis to separate DNA fragments
3. Southern blot and nucleic acid hybridisation using specific radioactive single-stranded probe that is complementary to part of the beta-globin gene
Visualise bands via autoradiography

Method 2:
Genomic DNA from an individual with sickle cell anemia and a normal individual are isolated
1. Conduct PCR by designing primers that flank beta-globin gene
2.Digest with restriciton enzyme, Mstll
Different sized restriction fragments are produced
3. Gel electrophoresis
Visualise bands using ethidium bromide and UV light

Question 26

VNTR: What does it indicate if the banding patterns of two DNA samples are very similar?

Answer 26

Since the number of repeats of a particular sequence is heritable, if the banding patterns of two DNA samples are very similar, it means they are from two very closely related individuals

Question 27

On what basis does RFLP in DNA fingerprinting work?

Answer 27

No two humans (except identical twins) have exactly the same genome
Tandem repeats of a particular DNA sequence occur in varying numbers in different individuals- enough to generate unique genetic profile for each individual

Question 28

Protocol of RFLP in DNA fingerprinting

Answer 28

Method 1:

1. Digest with restriciton enzymes that cut on either side of a tandem repeat loci
2. Separation of restriction fragments by gel electrophoresis
3. Incubate with radioactive probes for tandem repeats
4. Visualise via autoradiography

Method 2:

1. Design multiple PCR primer pairs which anneal to flanking sequence of different tandem repeats loci
2. Amplification of multiple tandem repeat loci
3. Separation of products by gel electrophoresis
4. Stain with ethidium bromide and visualise under UV light