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Biology A2 Rafa > 6.3 Manipulating Genomes > Flashcards

6.3 Manipulating Genomes Flashcards

1
Q

Define polymerase chain reaction

A

A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies

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2
Q

Applications of PCR

A 
Tissue typing
Detection of oncogenes
Detecting mutations 
Identifying viral infections
Monitoring the spread of infectious disease
Forensic science
Research
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3
Q

Applications of PCR

A 
Tissue typing
Detection of oncogenes
Detecting mutations 
Identifying viral infections
Monitoring the spread of infectious disease
Forensic science
Research
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4
Q

Define DNA ligase

A

Enzymes that catalyses the joining of sugar and phosphate groups writhing DNA

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5
Q

Define 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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6
Q

Define plasmids

A

Small loops of DNA in prokaryotic cells

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7
Q

Define 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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8
Q

Define restriction enzymes

A

Endonuclease enzymes that cleave DNA molecules at recognition sites

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9
Q

Define vector

A

In gene technology, anything that can carry/ insert DNA into a host organism; examples of such vectors include plasmids, viruses and certain bacteria

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10
Q

Necessary stages of genetic engineering

A
  • required gene is obtained
  • copy of the gene is placed inside a vector
  • vector carries gene to recipient cell
  • the recipient expresses the novel gene
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11
Q

What is DNA sequencing?

A

A technique that allows genes to be isolated and read

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12
Q

Who developed the technique that allows you to sequence whole genomes?

A

Fred Sanger

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13
Q

What was sangers approach?

A

Use a single strand of DNA as a template for four experiments in different dishes

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14
Q

What was in each one of Sanger’s dishes?

A

Solution with four bases and DNA polymerase

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15
Q

What did Sanger add to each tube?

A

Modified version of one of the DNA bases that meant once it was added to complementary DNA, no more bases could be added

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16
Q

What is each base labelled with in Sanger’s experiment?

A

Radioactive isotope

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17
Q

In gel electrophoresis, which fragments travel the furthest?

A

Smaller ones

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18
Q

What were the terminal bases labelled with in the first DNA sequencing machine?

A

Fluorescent dyes which glowed when scanned with a laser beam

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19
Q

What does pyrosequencing involve?

A

Synthesising a single strand of DNA, complementary to the one being sequences, one base at a time whilst detecting by light emission which base was added at each step

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20
Q

Steps of pyrosequencing

A

-DNA is cut into 300-800 base fragments using a nebuliser
-fragments degraded to single stranded DNA
-they are immobilised
-primer added and DNA incubated with enzymes
-activated nucleotides added and
light detected

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21
Q

In pyrosequencing, what enzymes is the DNA incubated with?

A

DNA polymerase, ATP sulfurylase, luciferase, apyrase and the substrates adenosine 5’ phosphosulfate (APS) and luciferin

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22
Q

What activated nucleotides are used in pyrosequencing?

A

ATP
TTP
CTP
GTP

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23
Q

What happens when an activated nucleotide is added in pyrosequencing?

A
  • Two extra phosphoryls are released as pyrophosphate (PPi)
  • The enzyme ATP sulfurylase converts pyrophosphate to ATP by combining it with APS
  • ATP and luciferin are converted to oxyluciferin by luciferase
  • this generates light
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24
Q

What are unincorporated nucleotides degraded by?

A

Apyrase

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25
Q

What are the applications of gene sequencing?

A
  • genome wide comparisons between individuals
  • genome wide comparisons between species
  • sequences of amino acids in polypeptides to be predicted
  • development of synthetic biology
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26
Q

Examples of synthetic biology applications?

A 
Information storage
Production of medicines
Novel proteins
Biosensors
Nanotechnology
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27
Q

Examples of nanotechnology

A

Amyloid fibres making biofilms

For functions such as adhesion

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28
Q

Basic stages of DNA profiling

A

Extract sample of DNA
DNA digested with restriction enzymes, which cut into fragments
Fragments separated by gel electrophoresis and stained
Banding pattern
Compare with other DNA

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29
Q

Why is restriction fragment length polymorphism analysis no longer used?

A

Laborious

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30
Q

What types of DNA are analysed in DNA profiling?

A

Short tandem repeat (STR) sequences of DNA

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31
Q

How many STRs are analysed simultaneously?

A

13

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32
Q

Applications of DNA profiling

A

Forensic science
Maternity and paternity disputes
Analysis of disease
Plant and animal breeding (reduces inbreeding)

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33
Q

How has profiling been used in forensic science?

A

Identifying nazi war criminals in South America
Identify remains of Romanov family
Victims bodies in disasters

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34
Q

How much of STR repeats come from mother and father?

A

Half from mother half from father

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35
Q

Example of how DNA profiling is used in disease analysis

A
  • detect haemoglobin present and aid diagnosis of sickle cell anaemia
  • Huntington’s repeat sequence number
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36
Q

What is PCR?

A

A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies

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37
Q

What does PCR do?

A

Amplify amount of DNA

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38
Q

What facts does PCR rely on?

A
  • DNA is anti parallel
  • have 5’ and 3’ end
  • DNA only grows from 3’ end
  • complementary bases pair up
39
Q

How does PCR differ from DNA replication?

A

Short sequence replicated, not chromosomes
Requires primer molecules
Cycle of heating and cooling need to separate DNA strands, bind primers to strands and for strands to be replicated

40
Q

What type of reaction is PCR?

A

Cyclic

41
Q

Why was the process of PCR slow at first and how was it sped up?

A
  • DNA was heated to denature it and then cooled to 35 degreees to anneal primers and allow polymerase to work
  • DNA polymerase was obtained from Thermophilus aquaticus, thermophilic bacterium
  • Taq polymerase is stable at high temperatures
42
Q

Applications of PCR?

A
  • tissue typing
  • detection of oncogenes
  • detecting mutations
  • identifying viral infections
  • ANALYSIS OF DISEASE RISK
  • FORENSICS
  • research
  • to produce enough DNA to mix with cut plasmids to insert into bacteria (in vivo cloning)
43
Q

What is tissue typing?

A

Donor and recipient tissues typed before transplantation to reduce risk of rejection

44
Q

What viral infections can PCR identify?

A

HIV or hepatitis C

45
Q

What’s the sample of DNA mixed with in PCR to start? + Where is this mixture put?

A 
DNA nucleotides
Primers
Magnesium ions
Taq DNA polymerase 
-All in thermocycler
46
Q

What is the DNA heated to the first time in PCR and why?

A

94-96 degrees
To break H bonds between complementary nucleotide bases
Two single strands of DNA

47
Q

What is the PCR mixture cooled to and why and what is produced?

A

68 degrees
Primers can anneal to one end of each single strand of DNA
A small section of double stranded DNA at the end of each single stranded molecule

48
Q

What is the optimum temp for Taq polymerase?

A

72 degrees and the mixture is raised to this

49
Q

What does Taq polymerase do?

A

Catalyses the added of nucleotides to single stranded DNA
Start at the end with primer
5’ to 3’

50
Q

How does the amount of DNA increase in PCR?

A

Exponentially

1-2-4-8-16-32-64-128

51
Q

After running gel electrophoresis on your DNA sequence what do you expose it to?

A

Photographic paper due to the radioactive isotopes

52
Q

Name of sequences forensic DNA tests use to identify individuals

A

Variable number tandem repeats

53
Q

Advantages of PCR?

A
  • rapid

- no living things involved

54
Q

Disadvantages of PCR?

A

Contaminant DNA may also be amplified

55
Q

What is electrophoresis?

A

process used to separate proteins or DNA fragments of different sizes

56
Q

What does electrophoresis use?

A

Agarose gel plate covered by a buffer solution

Electrodes at each end

57
Q

What is the overall charge of DNA and why?

A

Negative due to its many phosphate groups

58
Q

Which way do DNA fragments move in gel electrophoresis?

A

Towards the positive electrode (anode)

59
Q

Why are the samples in electrophoresis only separated by size?

A

Because different size fragments of DNA all have a similar surface charge

60
Q

Which fragments of DNA move the furthest in gel electrophoresis?

A

The shortest ones

61
Q

Difference between electrophoresis of proteins and electrophoresis of DNA?

A

-proteins = often carried out in the presence of a charged detergent such as sodium dodecyl sulfate (SDS) which equalises surface charge

62
Q

Why do we need SDS in protein electrophoresis?

A

Because proteins may have different surface charge

63
Q

What can gel electrophoresis of proteins be used to analyse?

A

types of haemoglobin for:

  • sickle cell anaemia
  • aplastic anaemia, thalassaemia and leukaemia
64
Q

What are DNA probes?

A

Small bits of DNA which know sequences
single stranded
tagges

65
Q

What can DNA probes be labelled using?

A
  • radioactive marker

- fluorescent marker (emits colour change on exposure to UV light)

66
Q

What can we use DNA probes for?

A
  • locating a gene to be used in genetic engineering
  • genome comparison studies
  • identifying alleles for genetic disease
67
Q

What is a DNA microarray?

A

A number of different probes on a fixed surface

68
Q

How do microarrays work?

A

You put your sample of DNA onto the plate with the probes and scan it

  • probes may be flourescently labelled and fluoresced when hybridised to samples of added DNA
  • tell you which probes were successful in detecting complementary sequences
  • can infer which sequences of DNA were in samples
69
Q

Use of microarrays and electrophoresis

A
  • locating and identifying alleles
  • genetic screening
  • personalised medicine
  • genetic counselling
70
Q

Define DNA ligase

A

enzyme that catalyses the joining of sugar and phosphate groups within DNA

71
Q

What is electroporation?

A

method for introducinga vector with a novel gene into a cell; a pulse of electricity makes the recipient cell more porous

72
Q

What are plasmids?

A

small loops of DNA in prokaryotic cells

73
Q

What is recombiant DNA?

A

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

74
Q

What are restriction enzymes?

A

endonuclease enzymes that cleave DNA molecules at specific recognition sites

75
Q

What is a vector?

A

in gene technology, anything that can carry/insert DNA into a host organism; examples of such vectors include plasmids, viruses and certain bacteria

76
Q

Necessary stages of genetic engineering

A
  • required gene obtained
  • copy placed into vector
  • vector carried gene into recipient cell
  • recipient expresses the novel gene
77
Q

Methods for obtaining required gene in genetic engineering

A
  • mRNA obtained, reverse transcriptase makes a single strand of complementary DNA (cDNA) with the addition of primers and DNA polymerase can make double stranded
  • automated polynucleotide synthesiser (if you know whole sequence)
  • PCR to amplify known gene
  • DNA probe locates gene and cut out using restriction enzymes
78
Q

How can you place a gene into a vector?

A
  • plasmids obtained from microorganisms and mix with restriction enzymes that cut plasmid at specific recognition sites
  • the plasmid now has exposed unpaired nucleotide bases, sticky ends
  • free nucleotide bases that match are added to the gene bing inserted and DNA ligase catalyses the annealing of the gene into plasmid
  • gene may be seal into a weakened virus that could carry it into a host cell
79
Q

Methods of putting vector into recipient cell

A

-heat shock treatment
-electroporation
-electrofusion
-transfection
T1 (recombinant) plasmids inserted into bacterium, Agrobacterium tumefaciens, which naturally inserts
-gene gun

80
Q

What is heat shock treatment?

A

bacteria subjects to alternating 0-42 degrees in the presence of calcium chloride
membranes become more porous, DNA can enter

81
Q

What is electroporation?

A

high voltage pulse disrupts membrane

82
Q

What is electrofusion?

A

electrical fields help introduce DNA into cells

83
Q

What is transfection?

A

DNA packaged into bacteriophage

transfects host cell

84
Q

What are direct methods of introducing genes into recipient?

A

If plants are not susceptible to A.tumefaciens
small pieces of gold or tungsten coated with DNA and shot into plant cells
GENE GUN

85
Q

Ethical considerations of genetic engineering?

A
  • escape of resistance genes (into weeds, eg herbicide resistance)
  • health and welfare of GM animals (testing)
86
Q

Positive of GM soya

A

resistant to herbicides

weeds competing with soya plants can be killed using herbicides

87
Q

Negative of GM soya

A

herbicide resistance pass into weeds producing superweeds

88
Q

Positive of GM pathogens

A
  • viruses, modified to having no virulence, make vaccines that don’t make people ill
  • used as vectors in gene therapy
89
Q

Negative of GM pathogens

A

-uses of viruses in gene therapy, allele may be inserted into genome in a way that increases risk of cancer or interferes with gene regulation

90
Q

Positive of GM animals to produce pharmaceuticals

A

-gene inserted into goats or sheep and they produce human protein in their milk

91
Q

Negative of GM animals to produce pharmaceuticals

A

welfare of GM goats and sheep

however, valuable and well looked after

92
Q

Issues relating to patenting and technology transfer

A

making genetically modified seed available to poor farmers

93
Q

difference between somatic and germ line gene therapy?

A

somatic can target specific tissues in need of treatment

germline cannot

Biology A2 Rafa (14 decks)

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