21 Manipulating Genomes

Question 1

What is a genome?

Answer 1

The genome of an organism is all of the genetic material it contains.

Question 2

What are the techniques for studying genes?

Answer 2

• The polymerase chain reaction
• Cutting out DNA fragments using restriction enzymes
• Gel electrophoresis

Question 3

What is PCR used for?

Answer 3

PCR is used to select a fragment of DNA and amplify it to copies.
- E.g in crime scene when only tiny amounts of DNA may be available.

Question 4

What is placed in the PCR machine?

Answer 4

• DNA sample to be amplified
• Excess of 4 nucleotides
• Small primer DNA sequences
• Enzyme DNA polymerase

Question 5

What happens in Step 1 of PCR?

Answer 5

STRANDS SEPERATED

• The temperature in the PCR machine is increased to 95°C for 30 seconds.
• This denatures the DNA by breaking the hydrogen bonds holding the DNA strands together so they separate.

Question 6

What happens in Step 2 of PCR?

Answer 6

ANNEALING OF THE PRIMERS

• The temperature is decreased to 55°C and the primers bind (anneal) to the ends of DNA strands.
• They are needed for the replication of the strands to occur.

Question 7

What happens in Step 3 of PCR?

Answer 7

SYNTHESIS OF DNA

• The temperature is increased to 72°C for at least one minute, this is the optimum temperature for DNA polymerase to work best.
• DNA polymerase adds bases to the primer, building up complementary strands of DNA and so producing double-stranded DNA identical to the original sequence.
• The enzyme Taq polymerase is used.

Question 8

As well as PCR, what is another way to get a DNA fragment?

Answer 8

Via restriction enzymes.

• Some sections of DNA have palindromic sequences of nucleotides.
• These sequences consist of antiparallel base pairs (read in opposite directions)
• Restriction enzymes recognise specific palindromic sequences and cut the DNA at these places.

Question 9

What are sticky ends?

Answer 9

Small tails of unpaired bases at each end of the fragment.

Question 10

Why are sticky ends useful?

Answer 10

Sticky ends can be used to anneal the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.

Question 11

What is electrophoresis?

Answer 11

• Electrophoresis is a technique that uses an electrical current to separate out DNA fragments.
• DNA fragments are put into wells in agarose gel strips which also contain a buffering solution to maintain constant pH.
• The gel is then placed in an alkaline solution to denature the DNA fragments.
• The two DNA strands of each fragment seperate.

Question 12

Why is electrophoresis used?

Answer 12

Electrophoresis forms the basis of DNA profiling- a process that can be used to determine how closely related two or more organisms are.

Question 13

What is DNA profiling?

Answer 13

DNA profiling produces an image of the patterns in non-coding DNA of an individual.

Question 14

What are the uses of DNA profiling?

Answer 14

1) FORENSIC SCIENCE: Forensic scientists use DNA profiling to compare samples of DNA collected from crime scenes to samples of DNA from possible suspects, to link them from crime scenes.
2) MEDICAL DIAGNOSIS: Can be used to analyse risk of genetic disorders.

Question 15

How is a DNA profile produced?

Answer 15

1) Extracting DNA- DNA extracted from tissue sample, or amplified using PCR
2) Digesting sample- Strands of DNA are cut into small fragments using restriction endonucleases. These cut DNA at a recognition side (specific nucleotide sequence)
3) Separating DNA fragments- Cut fragments of DNA are separated using gel electrophoresis. DNA fragments are transferred from the gel to nylon membrane via Southern blotting.
4) Hybridisation- Radioactive or fluorescent DNA probes are added in excess to DNA fragments on the membrane. DNA probes are short DNA sequences complementary to the sequences.
5) If Radioactive labels added- X-ray images taken
If fluorescent labels added- Membrane placed under UV light so fluorescent tags show.
The fragments give a pattern of bars- DNA profile.

Question 16

What is computational biology?

Answer 16

The study of biology using computational techniques to analyse large amounts of data.

Question 17

What is bioinformatics?

Answer 17

• The development of software and computing tools needed to analyse and organise raw biological data.
• Computational biology then uses this data to build theoretical models of biological systems which predicts what will happen in different circumstances.

Question 18

What is genomics?

Answer 18

The field of genetics that applies DNA sequencing methods and computational biology to analyse the structure and function of genomes.

Question 19

What are the uses of genome sequencing?

Answer 19

• Analysing the human genome
• Analysing the genomes of pathogens
• Identifying species
• Searching for evolutionary relationships

Question 20

What are the benefits of analysing the human genome?

Answer 20

• Computers can analyse and compare the genomes of many individuals, revealing patterns in the DNA we inherit and the diseases to which we are vulnerable.

Question 21

What are benefits of analysing the genomes of pathogens?

Answer 21

• Can find out the source of an infection
• Can identify antibiotic-resistant strains of bacteria and plan suitable treatment options.
• Can track the progress of an outbreak of a potentially serious disease and monitor potential epidemics
• Can identify regions in the genome of pathogens that may be useful targets in the development of new drugs and to identify genetic markers for use in vaccines.

Question 22

What is DNA barcoding?

Answer 22

• When scientists identify species using relatively short sections of DNA from a conserved region of the genome.
• This section is small enough to be sequenced quickly and cheaply, yet varies enough to give clear differences between species.

Question 23

What is proteomics?

Answer 23

Proteomics is the study and amino acid sequencing of an organisms entire protein complement.

Question 24

What are spliceosomes?

Answer 24

• Spliceosomes are enzyme complexes that join exons together to give the mature functional mRNA.
• The spliceosomes may join the same exons in a variety of ways.
• As a result, a single gene may produce several versions of functional mRNA, which in turn codes for different arrangements of amino acids, giving different proteins, resulting in several different phenotypes.

Question 25

What is synthetic biology?

Answer 25

The design and construction of novel biological pathways, organisms or devices, or the redesign of existing natural biological systems.

Question 26

What are the techniques of synthetic biology?

Answer 26

- Genetic engineering - Use of biological systems in industrial contexts e.g production of drugs from microorganisms - Synthesis of new genes to replace faulty genes - Synthesis of a new organism

Question 27

How is DNA sequencing carried out?

Answer 27

1) The DNA for sequencing is mixed with a primer, DNA polymerase, an excess of normal nucleotides (containing bases A, T, C and G) and terminator bases. 2) The mixture is placed in a thermal cycler, and at 96°C the double stranded DNA separates into single strands, at 50°C the primers anneal to the DNA strand. 3) At 60°C DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to the single strand DNA template. 4) Each time a terminator base is incorporated instead of a normal nucleotide, the synthesis of DNA is terminated as no more bases can be added. - As the chain-terminating bases are present in lower amounts and are added at random, this results in many DNA fragments of different lengths depending on where the chain terminating bases have been added during the process. - After many cycles, all of the possible DNA chains will be produced with the reaction stopped at every base. - The DNA fragments are separated according to their length by capillary sequencing, which works like gel electrophoresis in minute capillary tubing. - The fluorescent markers on the terminator bases are used to identify the final base on each fragment. - Lasers detect the different colours and thus the orders of the sequence. 5) The order of bases in the capillary tubes shows the sequence of the new, complementary strand of DNA which has been made. - This is used to build up the sequence of the original DNA strand. - The data from the sequencing process is fed into a computer that reassembles the genomes by comparing all the fragments and finding the areas of overlap between them. - Once a genome is assembled, scientists identify the genes that code for specific characteristics.

Question 28

What is next generation sequencing?

Answer 28

When all of the clusters are being sequenced and imaged at the same time.

Question 29

What are the basic principles of genetic engineering?

Answer 29

Isolating a gene for a desirable characteristic in one organism and placing it into another organism, using a suitable vector.

Question 30

What does transgenic/genetically modified organism mean?

Answer 30

An organism that carries a gene from another organism

Question 31

What is the first stage of genetic engineering?

Answer 31

Isolating the desired gene. - Enzymes called restriction endonucleases cut the required gene from the DNA of an organism. It cuts the two DNA strands unevenly producing sticky ends. The sticky ends make it much easier to insert the desired gene into the DNA of a different organism. - Another technique involves isolating the mRNA for the desired gene and using the enzyme reverse transcriptase to convert mRNA go gene to cDNA (more time efficient as introns have been removed, already modified)

Question 32

What is the most commonly used vector in genetic engineering?

Answer 32

Plasmids- small circular molecules of DNA separate from the chromosomal DNA that can replicate independently.

Question 33

What occurs once a plasmid gets into a new host cell?

Answer 33

It combines with the host DNA to form recombinant DNA.

Question 34

How is a DNA fragment inserted into a plasmid?

Answer 34

1) The same restriction endonuclease used to isolate the DNA fragment is used to cut the plasmid. 2) This results in the plasmid having complementary sticky ends to the sticky ends of the DNA fragment. 3) Once the complementary bases of the two sticky ends are lined up, the enzyme DNA ligase forms phosphodiester bonds between the sugar and phosphate groups on the two strands of DNA, joining them together. 4) The plasmids used as vectors are usually given a second marker gene, which is used to show that the plasmid contains the recombinant gene.

Question 35

How is the plasmid with the recombinant DNA transferred into the host cell?

Answer 35

There are two ways: 1) The bacterial cells and plasmids are cultured in a calcium rich solution and the temperature is increased. This causes the bacterial membrane to become permeable and the plasmids can enter. 2) Electroporation: The use of a very tiny electric current to transfer genetically engineered plasmids into bacteria by making the membrane very porous. It is also used to get DNA fragments directly into eukaryotic cells.

Question 36

What is another way of producing genetically modified cells?

Answer 36

Electrofusion. - In electrofusion, tiny electric currents are applied to the membranes of two different cells. - This fuses the cell and nuclear membranes of the two different cells together to form a hybrid or polyploid cell containing DNA from both.

Question 37

How is insect resistance brought about in soya beans?

Answer 37

- Scientists inserted a gene into soya beans so they produce the Bt protein. - This protein is toxic to many pest insects that attack the plant.

Question 38

What are the pros of pest resistance in GM crops?

Answer 38

- Reduce the amount of pesticide spraying, protecting the environment and helping poor farmers. - Increased yield

Question 39

What are the cons of pest resistance in GM crops?

Answer 39

- Non-pest insects might be damaged by the toxins in the GM plants. - Insect pests may become resistant to the pesticides in GM crops.

Question 40

What are the pros of disease resistance in GM crops?

Answer 40

- Reduces crop losses and increases yield

Question 41

What are the cons of disease resistance in GM crops?

Answer 41

- Transferred genes might spread to wild populations and cause issues e.g superweeds

Question 42

What are the pros of herbicide resistance in GM crops?

Answer 42

- Herbicides can be used to reduce competing weeds and increase yields.

Question 43

What are the cons of herbicide resistance in GM crops?

Answer 43

- Biodiversity could be reduced if herbicides are overused to destroy weeds.

Question 44

What is the issue with patenting and technology transfer?

Answer 44

- When someone discovers a new technique and legally patents it, it means no one else can use it without payment. - The people who need benefits of drought/resistant crops and high yields the most might be unable to afford the GM seed, especially if they're in a less economically developed country.

Question 45

What is pharming?

Answer 45

- The use of genetically modified animals to produce pharmaceuticals.

Question 46

What are two aspects of pharming?

Answer 46

- Creating animal models: adding or removing genes so that animals develop certain diseases, acting as models for development of new therapies - Creating human proteins- animals are used to produce milk containing human proteins that can be harvested.

Question 47

What are the ethical issues with researching microorganisms?

Answer 47

- Some people are worried that scientists researching the pathogen could become infected with the live pathogen and potentially cause a mass outbreak of disease. - The genetically modified version of a pathogen could revert back to its original form and cause an outbreak of disease.

Question 48

What is somatic cell gene therapy?

Answer 48

Replacing a faulty gene with a healthy allele in affected somatic cells.

Question 49

What is germ line cell gene therapy?

Answer 49

Inserting a healthy allele into the germ cells or into a very early embryo.