Biology > Manipulating Genomes > Flashcards

Manipulating Genomes Flashcards

(52 cards)

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1
Q

What is PCR (Polymerase Chain Reaction)?

A

Also known as thermal cycling
Allows sections of DNA (up to ~4KB) to be copies many times
Quick and inexpensive

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2
Q

What properties of DNA does PCR rely on?

A

If heated, strands break into two separate single strandes (breaking H bonds)
DNA polymerase can add free nucleotides to 3’ (OH) end of DNA template
Complementary bases will naturally anneal forming H bonds

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3
Q

What are primers?

A

Short, single stranded sections of DNA
Complementary to one end of a single strand of DNA
Anneal to DNA by H bonds
Allows DNA polymerase to attach and begin to make a complementary strand of DNA
Sometimes known as oligonucleotides

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4
Q

What is needed to undergo PCR?

A

Taq DNA polymerase (from thermophillic bacteria: can withstand high temperatures
Primers
DNA to be copied
DNA nucleotides

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5
Q

What is the denaturing stage of PCR?

A

Reaction mixture is heated to 90°c-95°c.
Separates double stranded DNA by breaking H bonds between complementary base pairs

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6
Q

What is the annealing stage of PCR?

A

Reaction mixture cooled to around 60°C, allowing the primer to anneal with the DNA template. Cooler temp. allows H bonds to form

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7
Q

What is the extension stage of PCR?

A

Reaction mixture is heated to 72°C. Allows Taq DNA polymerase to synthesise new strand of DNA
Polymerase recognises the primer as the starting point for synthesis
Puts free floating nucleotides into the correct places along the DNA template to extend from the primer

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8
Q

What are the stages of PCR?

A
  1. DNA of gene to be copied is extracted
  2. Denaturing
  3. Annealing
  4. Extension
  5. Genes that was copies forms part of two new DNA molecules and when heated provides four template strands
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9
Q

What are restriction enzymes?

A

Cut DNA at specific points, specfic enzymes with cut at restriction site
If you know base sequence near ends of gene can use it to cut out gene of interest
Restriction enzymes cut DNA to leave ‘sticky ends’, naturally anneal with complementary base sequence

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10
Q

What is recombinant DNA?

A

DNA constructed in a laboratory by combining genetic material from two or more different sources, creating new DNA sequences not found naturally, often by inserting a desired gene into a carrier DNA (vector like a plasmid) from another organism

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11
Q

What steps are involved in removing a section of DNA and forming recombinant DNA?

A

Donor DNA: cut donor (target) DNA using specific restriction enzymes
Cut host DNA (plasmid or other vector) using same restriction enzyme
Joining: use enzyme ligase to join the DNA backbone of the target DNA to that of the vector

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12
Q

What are exons?

A

Genes that code for proteins
Around 2% of total DNA

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13
Q

What are introns?

A

Large areas of non-coding DNA
Removed from mRNA before translation into a protein

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14
Q

What is satellite DNA?

A

Sequences of DNA in introns
Repeated over and over again
Always occur in same position in chromosomes
Actual number of repeats in each satellite varies between us greatly
Inherit different lengths of repeats from both parents
This DNA that is used in DNA profiling due to variation of length

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15
Q

What are the steps to produce a DNA profile?

A
  1. DNA obtained from individual
  2. DNA digested with restriction enzymes, cut DNA at specific sites, cut into fragments: vary in size from person to person
  3. Fragment separated by gel electrophoresis and stained, larger fragments travel shortest distance
  4. Banding pattern can be seen
  5. DNA to which individual being compares treated in same way
  6. Banding patterns compares
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16
Q

How is DNA profiling used in forensic science?

A

Criminal investigations: PCR and DNA profiling performed on traces of DNA (blood, semen, saliva etc)
Profile compared to that taken from suspect, or DNA database
Provides evidence for guilt/innocence

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17
Q

How is DNA profiling used for paternity testing?

A

Can be used when paternity in doubt

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18
Q

How is DNA profiling used to identify individuals at risk of disease?

A

Certain non coding microsatellites associated with increased risk of particular diseases (eg cancer, heart disease) can be observed in DNA profiles

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19
Q

When is southern blotting used?

A

Used when wanting to identify a specific gene or fragment of DNA

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20
Q

What is the process of Southern Blotting?

A

Cut DNA with restriction enzymes
Run DNA fragments on gel
Blot fragments onto nitrocellulose filter
Heat filter to break H bonds and separate strands
Add probles (labelled with radioactive label/fluorescent dye)
Probe binds to complementary strand, view under X Ray film/UV light

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21
Q

What are probes?

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A

Short, single stranded sections of DNA which are complementary to the DNA you are looking for
Probes can be labelled/tagged with a radioactive marker or a fluorescent marker

22
Q

Where may probes be used?

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A

Locate a specific gene in genetic engineering
Identify same gene in variety of genomes from different species when conducting gene comparison studies
Identify the presence/absence of a specific allele for a specific genetic disease

23
Q

What are the principles of genetic engineering?

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A

Required gene obtained
Copy of gene placed into vector
Vector carries gene into recipient cell
Recipient cell expresses novel gene

24
Q

What is electrophoresis?

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A

A process used to separate protein or DNA fragments according to size, mass or charge

25
What is the process of genetically engineering bacteria?
1. Cut gene out of the genome using restriction enzymes 2. Make required gene from mRNA 3. Copy of gene placed into a vector 4. Vector carries gene into recipient cell 5. Recipient cell expresses novel gene
26
What is transformation?
Process whereby which the new gene is taken up by the recipient cell
27
What methods are used to get the plasmid through the bacterial membrane?
Heat shock treatment Electroporation
28
What is heat shock treatment?
Bacteria subjected to periods of 0°C and 45°C in the presence of calcium ions, making membrane more porous
29
What is electroporation?
High voltage pulse applied to the cell to disrupt membranes In prokaryotes, plasmid enters cytoplasm In eukaryotes, new DNA will cross both the plasma and nuclear membranes and fuse with nuclear DNA
30
What is replica plating?
1. Sample of bacteria, treated with transgenic plasmids, spread on master plate containing ampicillin. Ampicillin resistant bacteria grow 2. Touch master plate bacterial colonies to sterile cloth on which bacteria can grow 3. Touch the tetracycline plate to the cloth with bacterial colonies 4. Incubate 5. Colonies that grow on the master plate but not on the tetracycline replica plate have taken up transgenic plasmid
31
What concerns do people have in using antibiotic resistance marker genes for selection of transformed bacteria?
Plasmids containing antibiotic resistance could be transformed to pathogenic bacteria Could infect humans and would be unable to kill them as they had antibiotic resistance
32
What alternative marker genes are commonly used instead of bacteria?
Marker genes that code for the synthesis of a protein which fluoresces under UV light Marker gene that codes for the synthesis of an enzyme that changes colour of particular medium
33
What are common vectors for genetically engineering animal cells?
Liposomes Viruses
34
What is the method of genetic engineering in plants?
1. Cut leaf 2. Expose leaf to bacteria carrying a weedkiller resistance gene and an antibiotic resistance gene. Allow bacteria to deliver the genes into the leaf cells 3. Expose leaf to an antibiotic to kill cells that lack the new genes. Wait for surviving, gene altered cells to multiply and form a clump (callus) 4. Allow callus to sprout shoots and roots 5. The plants are transferred to soil where they can develop into fully differentiated adult plants that are resistant
35
What bacterium is used for plant cells?
Agrobacterium tumefaciens
36
What is DNA sequencing?
Process by which the precise order of a DNA molecule can be determined
37
What processes are used in DNA sequencing?
PCR DNA heated to denature and make single stranded Primer anneals to template strand Normal nucleotides and terminator nucleotides incorporated Chain elongation Separation of new fragments by electrophoresis
38
What are the ingredients of DNA sequencing?
DNA template to be sequenced (single strand) DNA polymerase Free nucleotides Terminator nucleotides
39
What is the process of DNA sequencing?
1. DNA template with primer attached is added to 4 separate reaction tubes (A,T,G,C) 2. DNA polymerase, normal nucleotides are also added 3. One type of terminator nucleotide is added to each of the 4 tubes 4. Free nucleotides will complementary base pair with template strand 5. DNA polymerase joins bases = chain elongation 6. When a terminator nucleotide is joined, chain elongation stops: terminator nucleotides are radioactively labelled (show up during gel electrophoresis) 7. Reaction is left long enough to ensure all possible fragments are formed in each tube 8. Each tube run on electrophoresis gel, can find complementary strand to template.
40
What is automated sequencing?
Each nucleotide lavelled with different fluorescent tag colour, electrophoresis through small tube where laser detects wavelength
41
What are the pros of pest resistant GM crops?
Reduces amount of pesticide spraying, protecting environment and helps poor farmers Increased yield
42
What are the pros of disease resistant GM crops?
Crop varieties resistant to common diseased can be produced, reducing crop losses and increases yield
43
What are the pros of herbicide resistant GM crops?
Herbicide can be used to reduce competing weeds, increases yield
44
What are the pros of extended shelf life GM crops?
Reduces food waste
45
What are the pros of growing conditions GM crops?
Crops can grow in wider range of conditions (flood/drought resistance)
46
What are the pros of nutritional value GM crops?
Can be increased, more vitamins
47
What are the pros of medical uses GM crops?
Plants can be used to produce human medicines/vaccines
48
What are the cons of pest resistant GM crops?
Pollen from crops could transfer to wild plants, transferring resitance gene, disrupting food chain Non-pest insects/predators of insects may be damaged by toxins Insects may become resistant
49
What are the cons of disease resistant GM crops?
Transferred genes may spread to wild populations and cause problems (superweeds)
50
What are the cons of herbicide resistant GM crops?
Biodiversity could be reduced if herbicides used to destroy weeds Superweeds
51
What are the cons of extended shelf life GM crops?
May reduce commercial demand for crop Nutritional value deteriorate
52
What are the cons of nutritional value GM crops?
People may be allergic to different proteins in GM crops
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