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A2 Biology > Manipulating Genomes > Flashcards

Manipulating Genomes Flashcards

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1
Q

Name four ways in which DNA can be manipulated

A
  • DNA profiling
  • Genomic sequencing
  • Genetic engineering
  • Gene therapy
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2
Q

In automated DNA sequencing, the reaction mixture contains what?

A
  • DNA Polymerase
  • Single stranded DNA fragment
  • Free DNA nucleotides (some have florescent markers)
  • Primers
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3
Q

What happens if florescent markers (on some of the free DNA nucleotides) are added to the chain?

A

DNA polymerase is thrown off and the strand cannot have anymore nucleotides

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4
Q

Outline the process of automated DNA sequencing

A
  1. Primer anneals at the 3’ end, allowing DNA polymerase to attach
  2. DNA polymerase adds free nucleotides, so the strand grows
  3. A modified nucleotide is added
  4. Enzyme is thrown off
  5. Reaction stops
  6. In every strand the final nucleotide has a specific colour
  7. As these strands are run through a machine, a laser reads the colour sequence
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5
Q

In automated DNA sequencing, what order do the strands move in when being read in a machine?

A
  • In order of increasing size
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6
Q

What is High throughput sequencing?

A

Methods of rapid, inexpensive gene sequencing developed in the 21st century

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7
Q

What is Pyrosequencing?

A

A type of high throughput sequencing developed in 1996

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8
Q

What does Pyrosequencing involve?

A

Involves synthesising a single strand of DNA complementary to the one being sequenced

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9
Q

Outline the process of Pyrosequencing

A
  1. DNA cut into fragments of 300-800 base pairs
  2. Fragments are degraded into single-strand
  3. Fragments incubated with primer, APS, Luciferin, and several enzymes, including DNA polymerase
  4. Nucleotides added are ATP, TTP, CTP, and GTP
  5. Nucleotides from chains complementary to the DNA fragments
  6. They dephosphorylate: i.e. ATP -> Adenosine; TTP -> Thymine etc
  7. APS + pyrophosphate —> ATP
  8. Lights is released in this reaction and detected by a camera
  9. Light patterns detected indicate amount of ATP and therefore DNA sequence
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10
Q

In a 10-hour run, how many bases are read in pyrosequencing?

A

400 million

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11
Q

Give 4 applications of DNA sequencing

A
  • Comparison between species
  • Comparisons between individuals
  • Predicting amino acid sequences
  • Synthetic biology
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12
Q

What can we now identify thanks to gene sequencing?

A

The presence of genes throughout many species

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13
Q

Give an example of a a gene found to be similar in two species thanks to gene sequencing - And what has been a consequence of this?

A

Pigs have similar gene for insulin as humans

- Therefore pig insulin used to be used to treat diabetes in humans

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14
Q

What does identifying genetic similarities help us do?

A

Track evolutionary paths/relationships of species

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15
Q

Where does the 0.1% difference DNA that makes humans different arise from?

A

Subsititutions

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16
Q

What is Epigenetics?

A

The study of how Methylation of DNA causes changes int he expression of some genes

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17
Q

Outline the process of DNA profiling

A
  1. DNA obtained from individual
  2. DNA cut by restriction enzymes
  3. Fragments are separated by gel electrophoresis
  4. Banding pattern can be seen
  5. Banding pattern compared to another individuals’ which has been treated with the same restriction enzymes
  6. Related individuals will have more similar banding patterns
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18
Q

How can DNA profiling be used in forensic science? (3)

A
  • Establish innocence of suspects
  • Identified Nazi war criminals hiding in south america
  • Identify victims body parts after air crashes/terror attacks
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19
Q

How can DNA profiling be used in the analysis of disease? (2)

A
  • Protein electrophoresis can identify type of haemoglobin produced by an individual
  • Diagnose sickle cell anaemia
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20
Q

What is Synthetic biology as an application of DNA sequencing? What can it produce?

A

It’s designing useful biological devices and systems

E.g. biomedicine, food production

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21
Q

Why is it helpful that we can predict amino acid sequences from DNA sequencing?

A
  • Because it’s easier to sequence DNA than polypeptides

- Can read amino acid sequence directly from DNA

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22
Q

What information is needed for predicting amino acid sequences?

A

What part of the DNA codes for introns/exons

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23
Q

What is PCR?

A

Polymerase Chain Reaction, is a technique used to amplify DNA which makes it more suitable for analysis. Millions of copies of the sample of DNA are made and the base sequence is retained

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24
Q

What are the four principles of PCR?

A
  • DNA consists of 2 antiparallel chains
  • Each strand has a 3’ end and a 5’ end (according to direction of phosphate backbone)
  • DNA only grows from the 3’ end
  • Complementary base pairs
25
Q

Name one problem with PCR

A

Any contamination will be amplified

26
Q

Give three uses of PCR

A
  • Cloning
  • Electrophoresis
  • Gene probes
27
Q

What is needed for the DNA replication in test tube phase of PCR?

A
  • DNA needs to be replicated
  • DNA Polymerase
  • Excess of A/C/T/G bases
  • Primers
28
Q

How are the DNA strands separated in the second step of PCR?

A

Heated to 95 degrees c

- Causes strands to separate

29
Q

What happens with primers in the 3rd step of PCR?

A

Cooled to 55 degrees c, primers are able to attach

30
Q

How is DNA replicated in the 4th step of PCR?

A
  • Heated to 72 degrees c

- DNA replication can now occur

31
Q

What happens in the 5th step of PCR?

A

The thermostable DNA polymerase is added

  • Taq polymerase is used
  • Bacteria that lives in deep, warm oceans
32
Q

Whats different between PCR and normal DNA replication?

A

Strand separation
- In the cell, DNA helices separates the DNA strands
- In PCR, heat and temperature do this
Primers
(Required to allow DNA Polymerase to begin)
- In cells, they are made by DNA Polymerase
- In PCR they are synthesised separately and added

33
Q

Outline the process of PCR

A
  1. Everything added (DNA Polymerase, Primers, Ex. bases)
  2. Heated to 95c
    • H bonds broken
  3. Cooled to 40c
    • Allows primers to join
  4. Increase temp to 72c
    • Allows DNA to replicate
  5. DNA replicated
  6. Heated again and process is repeated
34
Q

What is Gel Electrophoresis?

A

It used electric currents through Agarose gel to separate DNA fragments according to size

35
Q

Outline the process of electrophoresis

A
  • DNA samples treated with restriction enzymes to cut them into fragments
  • DNA samples placed into wells of the gel
  • Gel is immersed in a tank of buffer solution
  • Phosphate groups make DNA negatively charged, so DNA diffuses through gel towards the positive electrode
  • Shorter lengths of DNA move faster, so move further
  • Position of fragments can be shown by a dye that stains DNA molecules
36
Q

What are DNA probes?

A

Short, single stranded pieces of DNA

37
Q

How are DNA probes used in Electrophoresis?

A
  • The fragments are complementary to the piece of DNA that is being investigated
  • Probe is labelled using radioactive marker that emits a colour upon exposure to UV light
  • Copies of probe will anneal to any complementary single strand
  • Probes can thus locate specific sequences
38
Q

Give three examples of specific sequences that can be located by DNA probes

A
  1. Locate a desired gene for genetic engineering
  2. Identify the same gene on different genomes
  3. Identify the presence of absence of an allele
39
Q

What can Genetic Engineering also be known as? (2)

A

Recombinant DNA technology or Genetic modification

40
Q

What is Genetic Engineering?

A

It involves the manipulation and combination of genes from different organisms

41
Q

Outline the four steps of genetic engineering

A
  1. Specific gene is isolated and copied
  2. Gene is places inside vector (E.g. Plasmid)
  3. Vector carries gene to target cell
  4. Recipient cell now has recombinant DNA and expresses newly acquired gene
42
Q

How is a required gene isolated in step one of genetic engineering?

A
  • mRNA coding for the specific gene is obtained
  • ## Reverse transcriptase forms a single strand of complementary DNA based off the mRNA
43
Q

How is a required gene isolated in step one of genetic engineering?

A
  • mRNA coding for the specific gene is obtained
  • Reverse transcriptase forms a single strand of complementary DNA based off the mRNA
  • Adding primers and DNA Polymerase catalyses the formation of double stranded DNA
  • Gene is synthesised
    - Via PCR if gene sequence is known
    - Via Automated polynucleotide synthesis if unknown
  • DNA probe used to locate gene
  • Gene isolated by restriction enzymes
44
Q

How is an isolated gene placed inside a vector in step two of Genetic engineering?

A
  1. Cut plasmid at specific site using restriction enzymes
  2. Cuts made by restriction enzymes match the ends of the isolated gene (complementary ends are ‘Sticky ends”)
  3. DNA Ligase catalyses insertion of gene into plasmid
  4. Gene can also be sealed in an unattended virus
45
Q

What four ways can a vector carry the gene to the target cell in genetic engineering?

A
  1. Heat shock
  2. Electroporation
  3. Electrofusion
  4. Transfection
46
Q

How does Heat shock work as a way of getting the gene to the target cell in genetic engineering?

A
  • Bacteria subject to fluctuating temperatures between 0c and 42c
  • Cell walls and cell membranes become more permeable
  • Recombinant DNA enters cell more easily
47
Q

How does Electroporation work as a way of getting the gene to the target cell in genetic engineering?

A

High voltage applied to cell and the membrane becomes more permeable

48
Q

How does Electrofusion work as a way of getting the gene to the target cell in genetic engineering?

A

Electrical fields cause DNA to enter cells

49
Q

How does Transfection work as a way of getting the gene to the target cell in genetic engineering?

A

Gene inserted into bacteriophage which infects bacterial cell

50
Q

Give an ethical benefit and hazard to genetically modifying golden rice

A

Benefit = GM rice contains beta carotene to reduce rates of blindness in children suffering from Vit A deficiency

Hazard = original concerns that new seeds would have to be bought every year, developed so farmers can re-sow

51
Q

Give an ethical benefit and hazard to genetically modifying Pathogens

A

Benefit = GM viruses have no harmful effect so can be used to make vaccines, and can help in gene therapy

Hazard = In gene therapy, gene modification can increase risk of cancer

52
Q

Give an ethical benefit and hazard to genetically modifying mice

A

Benefit = GM mice used for research, E.g. breast and prostate cancer

Hazard = Welfare concerns

53
Q

Give an ethical benefit and hazard to genetically modifying Microorganisms

A

Benefit = GM E.coli makes insulin to treat diabetics

Hazard = GM microorganisms must be carefully contained. If they’re released into the wild their genes will spread and effect entire ecosystems

54
Q

What is Gene Therapy?

A

Gene therapy is the use of been technology to treat genetic disorders

(Usually involves adding a functional copy of gene to cells which only contain a dysfunctional copy)

55
Q

Outline Germ line cell therapy (A type of gene therapy)

Where is it illegal?

A
  • Gene engineered into sperm or egg
  • Ensures all cells int he adult organism will have a copy
  • Illegal in humans
56
Q

Outline Somatic Cell Gene Therapy (A type of gene therapy)

A
  • During growth, cells become specialised
  • In these specialised cells, genes are switched on and off
  • Although it has full genome, not all of it is active
  • Somatic Cell Gene Therapy adds or kills genes
57
Q

How does adding genes in Somatic cell Gene Therapy treat genetic disorders?

A
  • Some genes are caused by faulty alleles
  • Engineering a functional copy of that gene into the cell means that the polypeptide can be produced
  • Functional gene can be produced
58
Q

How does killing genes in Somatic Cell Gene Therapy treat genetic disorders?

A
  • Cancerous cells can be made to express certain genes and create proteins
  • These can make the cells vulnerable to attack by the immune system
  • = Targeted cancer treatments

A2 Biology (13 decks)

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