Manipulating Genomes

Question 1

What is Genetic Engineering?

Answer 1

the manipulation of an organism’s DNA, which normally involves extracting a gene from one organism and inserting it into another organism.

Question 2

What are transformed organisms/genetically engineered organisms?

Answer 2

organisms that have had their DNA altered by genetic engineering

Question 3

What is a transgenic organism?

Answer 3

an organism that has been genetically engineered to include a gene from a different species

Question 4

How is a DNA fragment (that contains the gene you want) isolated?

Answer 4

using restriction enzymes

Question 5

What are two examples of vectors in genetic engineering?

Answer 5

plasmids, bacteriophages (viruses that infect bacteria)

Question 6

What is ligation?

Answer 6

when DNA ligase joins up the sugar-phosphate backbone of DNA fragments and vector DNA

Question 7

What is recombinant DNA?

Answer 7

the new combination of bases in the DNA (vector DNA + DNA fragments) following ligation

Question 8

What is the electroporation technique?

Answer 8

• when a suspension of the bacterial cells (containing recombinant DNA), is mixed with the plasmid vector and placed into an electroporator
• an electrical field is created in the mixture when switched on, which increases the permeability of the bacterial cell membranes and allows them to take in the plasmids

Question 9

What (3) techniques are used to study genes and their functions?

Answer 9

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

Question 10

What can PCR be used for?

Answer 10

to select a fragment of DNA and amplify it to produce millions of copies in a few hours

Question 11

Describe the stages of PCR

Answer 11

1) a DNA sample, free nucleotides, primers and DNA polymerase are mixed together.

2) the DNA mixture is heated to 95oC (to break the hydrogen bonds between two strands of DNA). DNA polymerase doesn’t denature even at this high temp (allows it to be recycled).

3) the mixture is cooled to between 50-65oC so primers can bind (anneal) to the strands.

4) the reaction mixture is heated to 72oC, so DNA polymerase can work.

5) the DNA polymerase lines up free DNA nucleotides alongside each template strand. Complementary base pairing means new complementary strands are formed.

6) two new copies of the fragment of DNA are formed and one cycle of PCR is complete.

7) the cycle starts again, with the mixture being heated to 95oC and this time all four strands (2 new + 2 original) are used as templates.

8) each PCR cycle doubles the amount of DNA.

Question 12

What is a primer?

Answer 12

a short piece of DNA that are complementary to the bases at the start of the fragment you want.

Question 13

What is DNA polymerase?

Answer 13

an enzyme that creates new DNA strands

Question 14

What is DNA sequencing used for?

Answer 14

to work out the sequence of bases in a section of DNA.

Question 15

What is high-throughput sequencing?

Answer 15

the name given to the range of methods which have allowed very rapid sequencing of DNA.

Question 16

What is next-generation sequencing?

Answer 16

the name given to the newest methods of rapid sequencing, which do not involve electrophoresis.

Question 17

What is the function of a restriction enzyme in genetic engineering?

Answer 17

to cut DNA at specific base sequences.

Question 18

What is the function of the ligase enzyme in genetic engineering?

Answer 18

joins the DNA/base to a plasmid/vector.

Question 19

What is used as a vehicle to transfer DNA into a host cell in genetic engineering?

Answer 19

a vector.

Question 20

What are 3 types of vector used in genetic engineering?

Answer 20

• plasmids
• viruses
• liposomes

Question 21

Describe the electrophoresis process.

Answer 21

1) add agarose gel to a gel tray and leave to solidify and then create a row of wells at one end.

2) put the gel tray into a gel box (with the wells closest to the negative electrode in the box).

3) add buffer solution to the reservoirs at the side of the gel box, so the surface of the gel becomes covered in it.

4) using a micropipette, add loading dye to each fragmented DNA sample in each well (being careful not to pierce the gel with the micropipette, and using a clean one for each well).

5) record the DNA sample added to each well.

6) put a lid on the gel box and connect leads to provide a power supply (e.g: 100V), causing an electrical current to be passed through the gel.

• DNA is negatively charged, so moves to the positive electrode (anode) of the gel.
  Small DNA fragments move faster and travel further, so can be separated according to size.

7) leave to run for 30mins before removing the gel tray, and tipping off the buffer solution.

8) wearing gloves, stain the DNA fragments and then rinse the gel with water, so the bands of different fragments are visible (the size is measured in bases).

Question 22

Why do scientists complete electrophoresis?

Answer 22

to separate out DNA fragments, RNA fragments or proteins according to their size.

Question 23

Why do scientists use loading dye in electrophoresis?

Answer 23

• it helps sample of DNA sink to the bottom of the wells.
• it makes the DNA easier to see.

Question 24

What must be done if electrophoresis is done on RNA fragments and why?

Answer 24

must mix with a chemical that denatures the proteins so they have the same charge, because they can be positively or negatively charged.

Question 25

What are restriction enzymes?

Answer 25

enzymes that recognise specific palindromic sequences (recognition sequences) and cut the DNA in these places.

Question 26

What are 'sticky ends' and what can they be used for?

Answer 26

small tails of unpaired bases at each end of a fragment. - they can be used to bind (anneal) the DNA fragments to another piece of DNA with complementary sequences of sticky ends.

Question 27

How can DNA profiling be used in forensic science?

Answer 27

- DNA is isolated from collected samples. - PCR is used to amplify. - PCR products are run on an electrophoresis gel and DNA profiles produced are compared to see if any match.

Question 28

How is genetic engineering carried out?

Answer 28

1) the DNA fragment containing the desired gene is obtained using restriction enzymes. 2) the DNA fragment is inserted into a vector using restriction enzymes and DNA ligase. 3) the vector transfers the gene into the bacteria.

Question 29

How is a DNA fragment inserted into a vector?

Answer 29

1) the vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment containing the desired gene, so the sticky ends of the vector are complementary to those of the DNA fragment containing the gene. 2) the vector DNA and DNA fragment are mixed together with DNA ligase (through ligation). 3) forms recombinant DNA.

Question 30

What are the ethical complications of genetic engineering of pathogens?

Answer 30

+ treat previously untreatable diseases. -could infect scientists researching, due to the use of live pathogens which could cause a mass outbreak of disease. -GM pathogen could revert back to its original form, and cause an outbreak. - engineer dangerous pathogens, to maliciously create agents for biowarfare.

Question 31

What are the 2 types of gene therapy?

Answer 31

- somatic therapy - germ line therapy

Question 32

What is somatic therapy?

Answer 32

this involves altering the alleles in body cells, particularly the cells that are most affected by the disorder.

Question 33

What is germ line therapy?

Answer 33

this involves altering the alleles in their sex cells, so every cell of any offspring produced from these cells will be affected by the gene therapy and they won't inherit the disease.

Question 34

What are the positive ethical issues of gene therapy?

Answer 34

- prolong lives for those with genetic disorders. - give people with genetic disorders a better quality of life. - carriers of a genetic disorder ma be able to conceive a baby without the disorder or risk of cancer (only in germ line therapy). - decrease the number of sufferers from the genetic disorder (only in germ line therapy).

Question 35

What are the negative ethical issues of gene therapy?

Answer 35

- technology could be used in other ways other than medical treatment (e.g: treat cosmetic ageing). - potential to do more harm than good when using the technology (e.g: risk over-expression of genes). - expensive (some believe that health resources could be better spent on other treatments that have passed clinical trials).

Question 36

What are disadvantages of gene therapy?

Answer 36

- short-lived effects of treatment (only in somatic therapy). - patients may have to undergo multiple treatments (only in somatic therapy). - may be difficult to get allele into specific body cells. - vectors may be identified as foreign by the body, eliciting an immune response. - an allele may be inserted into the wrong place in the DNA, causing more problems. - an inserted allele could get over-expressed, producing too much of the missing protein.

Question 37

Describe the chain-termination method.

Answer 37

1) a mixture containing: - a single-stranded DNA template - DNA primer - DNA polymerase - free nucleotides - fluorescently-labelled modified nucleotide are added to 4 separate tubes. 2) the tubes undergo PCR, producing many strands of DNA of different lengths because each one terminates at a different point depending on where the modified nucleotide was added. 3) the DNA fragments in each tube are separated by electrophoresis and visualised under UV light (due to florescent label). 4) the complementary base sequence can be read from the gel, with the smallest nucleotide at the bottom. Each band after this represents one more base added (read bottom up to get DNA sequence).

Question 38

What must be done when sequencing a whole genome using the chain-termination method?

Answer 38

- genome is cut into smaller fragments using restriction enzymes. - fragments are into bacterial artificial chromosomes (each with an individual one). - these BACs are inserted into bacteria. - the bacteria divide, creating colonies of clones cells that all contain a specific DNA fragment (the colonies make up a complete genomic DNA library). - DNA is extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNA. - each piece of DNA is sequenced, using the chain-terminating method, and put back in order to give the full sequence from that BAC (using computer systems). - all BAC DNA fragments are put in order by computers, to complete the entire genome.

Question 39

What does synthetic biology include?

Answer 39

- building biological systems from artificially made molecules to see if they work how we believe. - redesigning biological systems to perform better and include new molecules. - designing new biological systems and molecules that don't exist in the natural world, but could be useful to humans.

Question 40

What is bioinformatics?

Answer 40

developing and using computer software that can analyse, organise and store biological data.

Question 41

What is computational biology?

Answer 41

using computers to study biology (e.g: create computer simulations and mathematical models).

Question 42

What is epidemiology?

Answer 42

the study of health and disease, which considers distribution of a disease, its causes and its effects.