Biology Module 6 > Chapter 3 Manipulating genomes > Flashcards

Chapter 3 Manipulating genomes Flashcards

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1
Q

What is germ line gene therapy?

A

Gene therapy by inserting functional alleles into gametes or zygotes

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2
Q

What is somatic cell gene therapy?

A

Gene therapy by inserting functional alleles into body cells

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3
Q

What are advantages and disadvantages of germ line gene therapy?

A

Advantage: Greater potential to reduce the number of people suffering with a genetic disorder as it can alter genetics of the individual and all of their future descendants.

Disadvantages: Concerns over consent and the potential increased risk of cancer.

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4
Q

What are common vectors for gene therapy?

A
  • Liposomes (spheres of lipid)
  • Plasmids
  • Viruses
  • Artificial chromosomes
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5
Q

What is gene therapy?

A

Inserting a functional allele of a particular gene into cells that contain only mutated and non-functioning alleles of that gene.

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6
Q

How do liposomes act as vectors for gene therapy?

A
  • Alleles can be packaged into liposomes, which are placed into an aerosol inhaler and sprayed into noses of patients.
  • Some will pass through the plasma membrane of cells lining the respiratory tract.
  • Some will then pass through the nuclear envelope and insert into the host genome.
  • The host cell will express the functional protein.
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7
Q

What gene do patients with cystic fibrosis lack?

A

A functioning CFTR gene

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8
Q

Why must gene therapy for cystic fibrosis be repeated regularly?

A

Epithelial cells lining the respiratory tract are replaced every 10-14 days, so this treatment has to be repeated at regular short intervals.

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9
Q

How do viruses act as vectors for gene therapy?

A

A virus is genetically modified to encase the functioning allele and is made unable to cause disease.

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10
Q

What are some potential problems to using viruses as vectors for gene therapy?

A
  • They could provoke an immune/ inflammatory response in the patient
  • The patient may become immune to the virus, making subsequent deliveries impossible
  • The virus may insert the allele into a genome in a location that disrupts a gene involved in regulating cell division, increasing the risk of cancer
  • The virus may insert the allele into a genome in a location that disrupts the regulation of the expression of other genes
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11
Q

How would artificial chromosomes act as vectors for gene therapy?

A

Genes would be inserted into an artificial chromosome that would co-exist with the other 46 chromosomes in target cells.

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12
Q

What is recombinant DNA?

A

A composite DNA molecule created in vitro by joining foreign DNA with a vector molecule such as a plasmid.

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13
Q

What are restriction enzymes?

A

Endonuclease enzymes that cleave DNA molecules at specific recognition sites.

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14
Q

What are sticky ends?

A

Short stretches of unpaired nucleotides/ bases, used for binding DNA from the gene of interest with the plasmid

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15
Q

What is electroporation?

A

Method for introducing a vector with a novel gene into a cell; a pulse of electricity makes the recipient cell membrane more porous.

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16
Q

What is DNA ligase?

A

Enzyme that catalyses the joining of sugar and phosphate groups within DNA.

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17
Q

What is a vector?

A

Anything that can carry/ insert into a host organism in gene technology

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18
Q

What is PCR?

A

Polymerase chain reaction - A technique used to amplify a short length of DNA (to make copies).

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19
Q

What is replica plating?

A

A technique that uses antibiotic resistance genes to determine if bacteria have successfully taken up recombinant plasmids.

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20
Q

What is genetic engineering?

A

It is also known as DNA recombinant technology or genetic modification and involves combining genetic material of of different organisms. Genes are isolated from one organism and inserted into another organism using suitable vectors.

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21
Q

What are the stages of genetic engineering?

A
  1. The required gene is obtained
  2. A copy of the gene is placed inside a vector
  3. The vector carries the gene into a recipient cell
  4. The recipient expresses the novel gene
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22
Q

How can you obtain the required gene in genetic engineering?

A
  • Reverse transcriptase catalyses the formation of a single strand of complementary DNA (cDNA) using mRNA from the required gene as a template. The addition of primers and DNA polymerase makes the cDNA into a double-stranded length of DNA, whose base sequence codes for the original protein.
  • Knowing the nucleotide sequence of the gene means it can be synthesised using an automated polynucleotide synthesiser.
  • Knowing the nucleotide sequence of the gene means they can design PCR primers to amplify the gene from the genome
  • A DNA probe can be used to locate a gene within the genome and the gene can then be cut out using restriction enzymes
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23
Q

How is a gene placed into a vector?

A
  • Plasmids from bacteria are mixed with restriction enzymes that cut the plasmid at specific recognition sites
  • The cut plasmid is exposed to sticky ends
  • If free nucleotide bases, complementary to the sticky ends of the plasmid are added to the end of the gene to be inserted, the gene and cut plasmid should anneal (bind) catalysed by DNA ligase.
  • A gene may also be sealed into an attenuated virus to be carried into a host cell.
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24
Q

How is a vector put into a recipient cell?

A
  • Heat shock treatment: bacteria are subjected to alternating periods of heat and cold in presence of calcium chloride, making their walls and membranes more porous
  • Electroporation: high voltage pulse is applied to the cell to disrupt the membrane
  • Electrofusion: electrical fields help to introduce DNA into cells
  • Transfection: DNA is packaged into a bacteriophage, which can then transfect the host cell
  • Recombinant plasmids are inserted into a bacterium (Agrobacterium tumefaciens) which infects some plants and naturally inserts its genome into host cell genome
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25
What is the direct method of introducing a gene into a recipient?
Gene gun - small pieces of gold/ tungsten are coated with DNA and shot into cells
26
How do we ensure that transgenic bacteria are not dangerous if released due to their antibiotic resistance?
They have a gene knocked out meaning they cannot synthesise a specific nutrient. They are given the nutrient in their growth medium, but will not survive outside of the laboratory.
27
What is the process of PCR?
1. DNA is mixed with DNA nucleotides, primers, magnesium ions and Taq polymerase 2. Solution is heated to 95°C to denature the double-stranded DNA into two single strands by breaking H bonds between bases 3. Solution is cooled to 65°C, allowing DNA primers to anneal/ bind 4. Temperature is raised to 72°C, keeping DNA as single strands 5. Taq polymerase enzyme binds to the end with the primer and catalyses the addition of DNA nucleotides, starting at the end with the primer and proceeding in the 5' to 3' direction until it reaches the other end of the molecule
28
How do primers bind to DNA?
Hydrogen bonds
29
What are some differences between PCR and DNA replication?
- PCR requires addition of primers, DNA replication doesn't - In PCR only short sequences of DNA can be replicated, but in DNA entire chromosomes are replicated - In PCR a cycle of heating and cooling is used, but in DNA replication temperature is constant - In PCR Taq polymerase is used, in DNA polymerase is used
30
What are some applications of PCR?
- Tissue typing: donor and recipient tissues can be typed before transplantation to reduce risk of infection - Genetic testing: detecting mutations/ oncogenes/ genetic diseases - Forensic science: small amounts of DNA are amplified to identify criminals - Identifying viral infection: sensitive PCR tests can detect small amounts of viral genome amongst host cells' DNA
31
What is the significance of Mg ions in PCR?
Mg is the cofactor for DNA polymerase
32
Where is Taq polymerase obtained from and what makes it suitable for PCR?
It is obtained from bacterium Thermus Aquaticus which lives in hot conditions. Taq polymerase is stable at high temperatures and it's optimum temperature is 72°C.
33
What is electrophoresis?
A technique used to separate different sized fragments of DNA
34
What are some applications of electrophoresis?
- Gene sequencing - DNA profiling/ fingerprinting - Determining if a recombinant organism has been produced
35
What is the equipment used for in electrophoresis and how is it set up?
An agarose gel plate is covered with a buffer inside an electrophoresis tank. Electrodes are placed at each end of the tank so when it is connected to a power supply an electric current passes through the gel. A comb is used to form wells for digested DNA to be added to on the negative end.
36
What happens to DNA before electrophoresis?
DNA is cut into fragments by restriction enzymes at 35°C - 40°C, (optimum temperature).
37
How do DNA move along the agarose plate?
DNA is negatively charged due to phosphate groups so moves from the negatively charged end to the positively charged end. Smaller fragments travel faster than larger fragments so travel further up the plate.
38
How are fragments made visible at the end of electrophoresis?
The buffer solution is poured away and a dye is added to the gel. The dye adheres to the DNA, staining the fragments.
39
How is electrophoresis used to separate proteins?
A detergent called SDS is used, which denatures proteins, allowing them to move through the gel as a rod-like structure. It is used to diagnose sickle cell anaemia, thalassemia and leukemia (where patients have more fetal haemoglobin than adult haemoglobin)
40
What are DNA probes?
A probe is a short, single strand of DNA that is complementary to the section of DNA being investigated and is used to identify/ locate genes.
41
How can DNA probes be labelled?
- A radioactive marker which darkens photographic film - A fluorescent marker which glows when exposed to UV light
42
What are microarrays?
A microarray consists of lots of probes on a fixed surface. Applying DNA to the surface can reveal presence of mutated alleles that match and anneal to complementary fixed probes. Before using a microarray DNA must be broken down into fragments and may be amplified using PCR.
43
What is DNA sequencing?
A technique that allows genes to be isolated and read to determine their precise nucleotide sequence.
44
What are single nucleotide polymorphisms (SNPs)?
Places on the DNA where substitutions can occur, resulting in different alleles. Places where our DNA is not shared and is unique to us.
45
How was DNA sequenced before the modern sequencing was established?
Sanger's approach - It used mRNA transcribed from the gene rather than the DNA. mRNA is unstable and this process was extremely slow and only suitable for very short genes (whole genomes couldn't be sequenced).
46
What are some applications of gene sequencing?
- comparison of DNA between different species - comparison of DNA between individuals of the same species - studying evolutionary relationships (DNA from bones of extinct animals can be amplified & sequenced) - predicting the sequence of amino acids and the structure/ function of the protein - synthetic biology
47
How is DNA cloned before sequencing?
It is first isolated using restriction enzymes. DNA is then inserted into a bacterial plasmid, which is then inserted into a bacterium host cell which divides, resulting in the plasmid with the DNA insert being copied many times.
48
How was the first DNA sequencing machine different from our modern machines?
Radioactive dyes rather than fluorescent dyes were used to label the terminal bases. The fluorescent dyes glowed when scanned with a laser beam and the light signature was identified by a computer so technicians no longer had to read autoradiograms, making it less labour intensive.
49
What 4 things are required for gene sequencing?
- DNA polymerase - DNA primers - Free nucleotide bases - Termination bases
50
What do termination bases do?
They prevent phosphodiester bonds forming so no more bases can be added to the sequence.
51
How do terminator bases allow us to identify the DNA sequence?
Terminator bases are coloured with fluorescent tags made visible by a laser. Each base has a different colour. The colour is detected by a camera and recorded.
52
What are some applications of synthetic biology?
- Production of medicines - Information storage - encodes vast amounts of DNA onto a single strand of synthetic DNA - Nanotechnology
53
What is the role of PCR in gene sequencing?
- to amplify & make many copies of DNA - makes copies that are a range of different lengths
54
What is the role of restriction enzymes in genetic engineering?
- cuts DNA at specific recognition sites into smaller fragments - cuts vectors/ plasmids to insert a functioning allele for genetic engineering/ gene therapy
55
Why must a genome be fragmented before sequencing?
- Whole genome is too large to be sequenced - Better accuracy/ fewer errors
56
What is the name of the most modern high throughput sequencing method?
Pyrosequencing
57
What is the process of pyrosequencing?
1. A long length of DNA is mechanically cut up into fragments of 300-800 base pairs using a nebuliser 2. The lengths are degraded to single stranded DNA which are immobilised 3. Sequencing primer is added 4. DNA is incubated with DNA polymerase, ATP sulfurylase, luciferin, luciferase, apyrase, APS (adenosine 5' phosphosulfate) and one of the activated nucleotides ATP, CTP GTP & TTP 4. One activated nucleotide, e.g. TTP, is incorporated into a complementary strand of DNA using the strand to be sequenced as a template 5. The 2 extra phosphoryls are released as pyrophosphate 6. In the presence of APS, ATP sulfurylase converts pyrophosphate into ATP 7. In the presence of this ATP, luciferase converts luciferin into oxyluciferin 8. This conversion creates visible light which is detected by a camera
58
What is the process of DNA profiling?
1. DNA is obtained from the individual from saliva, blood, hair or bones of ancient species 2. DNA is digested with restriction enzymes 3. Fragments are separated by gel electrophoresis & stained to produce bands that can be compared.
59
Applications of DNA profiling?
- Forensic science - Maternity/ Paternity disputes - Analysis of disease
60
What is the function of apyrase in pyrosequencing?
Apyrase degrades unincorporated (unused) activated nucleotide
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