Gene technologies (techniques)

Question 1

Which areas in science have benefited from our increased understanding of DNA?

Answer 1

• DNA profiling - used in forensics to identify potential suspects and also in maternity/paternity tests.
• Genomic sequencing and comparative genome mapping - used to gain greater understanding of functions of genes and regulators in DNA.
• Genetic engineering - Used to produce pharmaceutical products (e.g. insulin), agricultural products and potential xenotransplantation donors.
• Gene technology - Can potentially be used to treat genetic diseases.

Question 2

What is gel electrophoresis used for?

Answer 2

Separating different lengths of DNA fragments. The technique is so accurate that lengths of DNA differing by 1 base pair (bp) can be separated.

Question 3

What are the steps involved in electrophoresis?

Answer 3

1. Restriction enzymes are used to fragment DNA into different length fragments.
2. Agarose gel is set in electrophoresis tray and samples are loaded into wells set at the cathode (negative electrode) end.
3. Gel covered with buffer solution and electric current passed through solution for set amount of time.
4. A loading dye is usually used to make the fragment bands visible at the end.

Question 4

What are the principles behind electrophoresis?

Answer 4

• Phosphate groups on DNA have -ve charge and are attracted to anode (positive electrode), so DNA fragments move towards it through gel.
• Different sized fragments move at different speeds through gel.
• The smaller the fragment, the quicker and further it moves from wells, the closer it will be to the anode end of gel at end of experiment.
• The bigger the fragment, the slower it moves and closer it will be to wells and cathode end of gel at end of experiment.

Question 5

What are the steps involved in the Southern Blotting technique?

Answer 5

1. Nylon or nitrocellulose sheet placed over gel, covered with paper towels, pressed and left overnight.
2. This lifts the bands of DNA fragments onto the sheet.

Question 6

How can the bands of DNA be made visible after transfer onto nylon sheet?

Answer 6

The DNA fragments could have been stained with a radioactive probe before electrophoresis. Then photographic film simply needs to be put onto sheet and bands will develop on film in their respective positions.

Question 7

What is a DNA probe?

Answer 7

A short single-stranded piece of DNA (50-80 nucleotides long) complementary to the section of DNA under investigation, with a relevant marker attached to make them visible.

Question 8

What are the 2 ways DNA probes can be labelled?

Answer 8

1. Using radioactive marker (e.g. P32 in the phosphoryl groups that make up sugar-phosphate backbone). Visible when photographic film placed over it.
2. Using fluorescent marker (e.g. GFP) which emits colour when exposed to UV light.

Question 9

What is the process of the probe binding to DNA?

Answer 9

The probe will bind to a specific sequence of DNA by complementary base pairing via hydrogen bonds, so long as the DNA being analysed is also single stranded. This allows these sequences to be located/detected. The process of the probe binding is called Annealing.

Question 10

Why does the probe have to be short?

Answer 10

To allow the probe to be specific to only certain nucleotide sequences. If the probes are too long, it may have sites complementary to more than one nucleotide sequence.

Question 11

What are DNA probes used for?

Answer 11

• To locate desired genes on strand of DNA.
• To identify the same genes in the genome of different species when conducting genomic comparison studies.
• To detect the faulty allele(s) responsible for genetic diseases.

Question 12

What does PCR stand for?

Answer 12

Polymerase Chain Reaction.

Question 13

What is PCR used for?

Answer 13

To quickly and efficiently amplify DNA present in only small amounts to amounts large enough to be studied.

Question 14

What are the stages involved in PCR?

Answer 14

1. Sample of DNA mixed with free DNA nucleotide, primers and DNA polymerase.
2. The mixture is heated to 95ºC, which breaks hydrogen bonds between strands in double stranded DNA so that strands separate and become single stranded.
3. Primers are added and temperature reduced to 55ºC, these join (anneal) onto specific sites on single strands to form double stranded sections and act as starting points for replicate strands to form from.
4. Temperature raised to 72ºC (optimum for DNA polymerase)
5. Polymerase binds to primer formed double stranded sections on template strands and begins adding free nucleotides. This extends replicate strands by complementary base pairing.
6. When DNA polymerase reaches end of template strands, new double stranded lengths of DNA are formed (from template and replicate strands).
7. The whole process is repeated with the new replicate strands now acting as template strands, hence the chain reaction.

Question 15

What properties of DNA does PCR depend on?

Answer 15

1. Antiparallel strands.
2. Each strand has a 5’ (prime) and 3’ end.
3. Strand only grows from 3’ end.
4. Complementary base pairing.

Question 16

What are the differences between PCR and natural DNA replication?

Answer 16

• PCR can only replicate relatively short sequences of DNA whereas natural replication is done for whole chromosomes.
• Primers are required for PCR but not natural replication.
• Heat is used to separate strands in PCR as opposed to DNA helicase.

Question 17

Why are primers required for PCR?

Answer 17

Because DNA polymerase only acts on double strands.

Question 18

Why are 2 different primers usually added?

Answer 18

• There are 2 different single-stranded DNA that derive from original double strand.
• These have different nucleotide sequences at beginning of strand.
• 2 different primers complementary to both start points need to be added in order for both strands to act as templates.

Question 19

What is the overall process of sequencing a genome?

Answer 19

1. The genes/genome is mapped so that it is clear which chromosome/part of chromosome they came from.
2. The genome is then sheared into smaller sections of around 100,000bp long. The is called ‘shotgun approach’.
3. These sections are put into Bacterial Artificial Chromosomes (BACs) and put onto E. Coli to be copied. These form a ‘clone library’.
4. When ready to sequence, the E. Coli containing the section is cultured and the fragment is cut out using restriction enzymes.
5. The section is cut into smaller fragments using different restriction enzymes (so that they overlap) and are separated by electrophoresis.
6. The smaller fragments are individually sequenced and a computer reassembles the overlapping fragments to form the sequence of the original section.
7. The sequences of the sections are reassembled according to the original genomic map to construct the whole genome.

Question 20

How is a fragment of DNA sequenced?

Answer 20

1. The double stranded DNA is heated in order to break it into 2 single strands.
2. 1 Primer complementary to the start of 1 single strand is added to the mixture, along with free DNA nucleotides modified DNA nucleotides and DNA polymerase.
3. Modified nucleotides do not have a functional 5’ end so additional nucleotides cannot join onto it. This results in the copied sequence being terminated when a modified nucleotide joins.
4. Each modified nucleotide of a certain base has a different coloured fluorescent dye attached.
5. The mixture is put into an automated PCR machine and left to run for several hours for thousands of copy strands to be generated.
6. Because the nucleotides join at random, it is very likely there will be fragments that were terminated at every possible length along the template strand as it was read (I.e. at every nucleotide beyond the initial primer).
7. The different length fragments are separated by capillary electrophoresis whereby the distance is fixed but time varies as the different sized fragments travel at different speeds through agarose gel.
8. A laser detector reads the colour of each band, which represent the modified base that terminated the sequence. The colour peaks can then be plotted along a time graph and the sequence of the copied strand of DNA can be determined.

Question 21

How is the sequence of the original template strand determined from the coloured peaks?

Answer 21

• The smallest fragment travels the quickest, so will be detected first. The colour of the first peak (reading left to right) on the time graph will therefore represent the first base after the primer, the second peak the second base etc.
• This method can be used to simply read off the base sequence coming after primer.
• The sequence of the primer is known so can be simply added to the beginning of the obtained to construct the full sequence of the copied strand.
• Complementary base pairing can then be used to determine the sequence of the template strand.

Question 22

What is the limitation of genomic sequencing?

Answer 22

Can only be done for small fragments of DNA at one time (around 750bp).

Question 23

What can genomic sequencing be used for?

Answer 23

• Comparative gene mapping.
• Genomes of different species can be compared in order to identify common/similar protein. This would suggest they are essential to life.
• Comparing the genome of different species allows us to determine evolutionary relationships. The more similar the genomes, the closer related they are to each other.
• Allows us to model the effects of mutations in humans by simulating mutation in another organism with similar gene. This helps us understand the cause of genetic diseases.
• Comparing differences in related pathogenic/non-pathogenic organisms allow us to identify key factors associated with disease and potential vaccine/drug targets.
• Comparing genome of individual to groups of people with certain diseases allow us to identify presence of disease-causing mutations and determine the chance of that individual also acquiring disease.