OCR Biology A Level > 6.1.3 Manipulating Genomes > Flashcards

6.1.3 Manipulating Genomes Flashcards

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1
Q

What does ‘PCR’ stand for?

A

Polymerase chain reaction

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2
Q

What is the genome?

A

All the DNA in the cells of an organism

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3
Q

Where is DNA found in animals and plants?

A

Nucleus and mitochondria of animals
Also the chloroplast in plants

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4
Q

What are DNA exons and what percentage of DNA are they?

A

Regions of coding DNA
2% of DNA

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5
Q

What are DNA introns and what percentage of DNA are they?

A

Regions of non-coding DNA
98% of DNA

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6
Q

What does PCR produce and what type of cloning is it?

A

Produces copies of a specifi piece of DNA
Is In vitro cloning

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7
Q

What equipment does PCR use?

A

A thermal cycler

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8
Q

What 4 substances are required in PCR?

A
  • The DNA being copied
  • Large amount of the 4 nucleotides containing the four bases (A,C,T,G)
  • Primers
  • Taq DNA polymerase
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9
Q

What is type of DNA Polymerase and what special property does this have?

A

Taq DNA polymerase from bacteria in hot springs that is not denatured at high temperatures

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10
Q

What is a primer?

A

A short single stranded DNA molecule that is complementary in sequence to the beginning and end target sequence

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11
Q

How long is a primer?

A

10 base pairs

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12
Q

What is the role of a primer?

A

Mark the beginning and the end of a target sequence
Allow for the attachment of enzymes or nucleotides
Keep the 2 DNA strands apart

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13
Q

Explain the four stages of PCR

A
  1. Denaturation: DNA heated to 95°C so hydrogen bonds between bases break
  2. Annealing: Cool to 50-60°C so primers bind to strands
  3. Polymerisation/elongation: Heat to 72°C so taq DNA polymerase can join nucleotides
  4. Two copies of original DNA fragment formed - repeated 30x times
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14
Q

Why is PCR called ‘chain reaction’?

A

Becasuse the number of molecules increases expoentially when PCR is run repeatedly (20-30x), like in a chain reaction.

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15
Q

What are minisatellites?

A

Sequences of 20-50 base pairs repeated from 50 to several hundred times (VNTRs)

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16
Q

What are microstaellites?

A

2-4 bases repeated only 5-20 times (STRs)

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17
Q

How do satellites enable DNA profiling?

A

Position of microsatellite + macrosatellite repeats on the chomrosome is the same, but the number of repeats varies between individuals
An image is produced to show these patterns, allowing comparison between individuals

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18
Q

Name the 7 stages of DNA profiling

A
  1. Extraction
  2. Digestion
  3. Separation
  4. Hybridisation
  5. Development
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19
Q

Explain the steps of extraction in DNA profiling (3)

A
  1. DNA extracted from tissue sample
  2. Protease enzume added to hydrolyse histone proteins DNA is wound around
  3. PCR used to give enough DNA
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20
Q

Explain the steps of digestion in DNA profiling (3)

A
  1. Restriction endonuclease enzymes used to cut DNA into small fragments
  2. DNA cut at specific nucleotide sequence sites (Recognition sites)
  3. Two cuts made, one though each DNA strand in the double helix
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21
Q

What is the role of restriction endonuclease enzymes?

A

Cut useful genes out of the DNA
They leave sticky ends (unpaired bases in DNA) that enable joining up to other complementary DNA

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22
Q

Explain the steps of separation in DNA profiling (4)

A
  1. Cut fragments seperated to form a clear and recognisable pattern
  2. Gel electrophoresis used
  3. Gel immersed in alkali solution that carries charge to separate DNA double strands into single strands + separates fragments
  4. Single strand fragments transferred to nylon membrane via southern blotting
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23
Q

DNA is colourless, so how is it able to be seen?

A

Stained or radioactive marker used and photographic film marked, producing an autoradiograph

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24
Q

How does DNA become separated during gel electrophoresis?

A
  • In an electrical field it moves towards the positive side of the gel
  • DNA fragment size affects how far it travels
  • Smaller pieces travel further
  • Larger pieces travel slower + lag behind
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25
Why is DNA negatively charged?
Contains phosphate groups in the sugar phosphate sugar backbone, which is negatively charged
26
Explain the steps of hybridisastion in DNA profiling (3)
- Radioactive (Phosphorous-32) or flourescent probes added in excess to DNA fragments on membrane - Probes consist of short DNA/RNA sequences complementary to known DNA sequence - Under particular pH + temperature, probes bind to complementary stands of DNA to visualise band/patterns and to identify microsatellites
27
What is DNA annealing used for? (3)
- Locating a specific gene for genetic engineering - Identifying the same gene in different species - Identifying presence or absence of allele for a genetic disease
28
Explain the steps of development in DNA profiling (3)
- Xray images of membrane taken if radioactive labels used - Membrane placed under UV light if fluorescent labels added - Fragments give a pattern of bars = DNA profile - Gives a unique pattern for every person (except identical siblings)
29
Give uses of DNA profiling (5)
- Used to identify suspect from DNA left at crime scence - Prove paternity of a child - Prove/disprove family relations in immigration cases - Identify which species an organism belongs to/show evolutionary relationships - Identify individuals at risk of developing certain diseases
30
Define DNA sequencing
Working out the nucleotide base sequence of genes that have not been studied before
31
Explain the 5 steps of setting up manual DNA sequencing
1. Setup 4 test tubes with single stranded DNA fragments to be sequenced 2. Add many free ACTG free DNA nucleotides 3. Add a small amount of Adenine dideoxynucleotides to tube 1, repeat for each nucleotide 4. Add primers to start DNA synthesis 5. Add DNA polymerase
32
What does a termination nucleotide/didexoynucleotide do?
Stop DNA polymerase from replicating DNA
33
How is DNA manual DNA sequencing read?
Sections in each test tube ran though capillary sequencing/gel electrophoresis
34
How are fragments seperated?
By size - smaller sections move faster to positive end
35
How are terminator bases able to be see
Using flourescent tags that are scanned by lasers to show order of sequence
36
Explain the 10 steps of DNA sequencing
1. DNA mixed with primers, DNA polymerase, excess nucleotides, terminator bases 2. Mixture undergoes PCR in thermal cycler 3. At 96°C strands seperate 4. At 50°C rimers anneal to DNA strand 5. At 60°C DNA polymerase builds up new DNA strand 6. When terminator bases incorporated DNA synthesis inhibited 7. Fragments of different lengths created 8. After many cycles all possible DNA chains produced 9. DNA fragments seperated by length via capillary sequencing 10. Sequence of DNA on complementary strand present in capillary strand
37
How is next generation DNA sequencing better compared to manual DNA sequencing?
Much faster + automated
38
How does next generation sequencing work? (5)
- Flow cell slides used - PCR replicates DNA fragments in situ - Clusters of identical DNA fragments made - Coloured terminator bases still used to stop reaction so image can be taken - Clusters sequenced + imaged at the same time
39
List developments that have led to increased DNA sequencing speed
- Swapping radioactive tags for flourescent tags - Next generatio sequencing - Massive parallel sequencing
40
What is DNA sequecning used for?
Genome-wide comparison between individuals and species
41
What is genetic engineering?
Manipulation of the genome using recombnant DNA technology, where DNA from one organism is palced into the DNA of another organism
42
Briefly explain the 4 steps of using recombinant DNA technology
1. Find required gene 2. Copy of gene placed into vector 3. Vector rakes gene into recipient cell 4. Recipient expresses the gene through protein synthesis
43
Define a transgenic organism
An organism that carries a gene from another organism
44
What is another way to describe a transgenic organism?
A genetically modified organism (GMO)
45
Why is genetically engineering done?
To improve an organism e.g. Make a plant resistant to herbicides To use an organism to make a product e.g. Hormones made by bacteria
46
What enzyme is used to isolate desired genes and what do they do?
Restriction endonuclease enzymes break DNA at specific base sequences
47
What are sticky ends?
Strands unevenly cut with regions that have unpaired, exposed bases
48
How can mRNA be isolated for the desired gene?
Using reverse transcriptase enzyme to produce a single strand of complementary DNA
49
What does reverse transcriptase do?
Make DNA from mRNA
50
How is a double stranded DNA produced by reverse transcriptase? (3)
1. Reverse transcriptase mixed with mRNA and free DNA nucelotides 2. Reverse transcriptase joins DNA nucleotides to make complementary DNA (DNAc) 3.DNA polymerase enzyme joins free nucleotides to cDNA to form double stranded DNA
51
Where is isolated DNA inserted into?
A vector
52
What are commonly used vectors for formation of recombinant DNA?
Bacterial plasmids
53
What are GM bacteria used for?
To make human proteins e.g. Insulin
54
What are the vectors plasmids and bacteriphages used for?
Putting DNA into bacteria
55
What is the vectors virus' and liposomes used for?
Putting DNA into animal and plant cells
56
Where is DNA extracted from in genetic engineering?
A donor cell
57
Where does restriction endonuclease enzaymes cut DNA at, and what does it leave?
Specific palindromes, leaving sticky ends
58
What enables DNA carrying vectors to be taken up by bacteria?
The vector's small size
59
What is important about the restriction endoculease enzyme used to cut the plasmid removed from the bacteria, and why?
- The same restriction endonuclease enzyme has to be used to cut the plamid - At the same recongition site so that donor DNA will have complementary sticky ends
60
How is the desired donor DNA section selected?
Using gel electrophoresis and gene probe
61
What is the function of DNA ligase and what is this process called?
Joins complementary sticky ends between donor gene and plasmid The process is called ligation
62
What bonds are formed between sticky ends?
Hydrogen bonds between complementary bases + phosphodiester bonds
63
What is recombinant DNA?
A plasmid containing select DNA from donor DNA
64
What processes are used to increase the amount of recombinant DNA taken up by treated bacteria?
- Calcium salts - Heat shock treatment - Electroporation - Electrofusion
65
What happens to bacteria that do take up recombinant DNA?
Cultured to make copies of donor gene and produced the desired protein
66
How does using bacteria cause increased production of recombinant DNA?
Bacteria divide by binary fission, where the plasmid is repicated nad passed to duaghter cells that are genetically modified so produced the desired protein
67
What are the two methods DNA can be extracted for use in genetic engineering?
Method 1: DNA extracted from donor cell nad restriction endonuclease enzymes cut DNA at specific palindromic sequences, leaving sticky ends Method 2: mRNA extracted from donor cell and treated with reverse transcriptase enzymes, makign single stranded tempalte DNA, treated with DNA polymerase to give double stranded DNA
68
Name 3 ways vectors are transfered into the bacteria?
- Bacteria + plasmids cultured in calcium rich solution and temperature increased - Electroporation (High voltage causes temporary gaps in membrane to form) - Electrofusion (Electric field around the cell helps DNA to enter)
69
What do genetic markers allow for in recombinant DNA?
The identification of bacteria that have successfully taken up he recombinant plasmid.
70
Explain the 4 steps of replica plating
1. Marker genes inserted for antibiotic resistance via genetic engineering 2. Restriction endonuclease enzymes used to insert insulin gene within marker gene 3. If insulin gene inserted successfuly, the marker gene for antibiotic resistance is no longer functional 4. Therefore the bacteria will be killed by this antiboitic, shwoing insulin was succesfully inserted
71
What else can be used as gene markers?
Fourescent marekrs found in jellfish that glow in UV light
72
Why might flourescent UV markers be used instead of antibiotic resistance markers?
Concerns over the development of antibiotic resistant bacteria
73
Where could GM microorganisms be exploited by the government?
Pathogens in biological warfare
74
How has golden rice been bio-fortified?
Contains the gene for Beta-caroene that is convereted into Vitamin A in the gut
75
Benefits and ethical concerns of golden rice (3 + 4)
**Benefits** - Reduces vitamin A deficiency in developing world - Reduces blindness - Reduces deaths from Vitmain A deficiency **Ethical Concerns** - Reduced genetic biodiversity - Creates clones that could be killed by one disease - Risk of hybridisation with wild rice - Seeds expensive
76
What is somatic cell gene therapy?
Replacing mutant alleles with healthy alleles in the affected somatic cells, usually usin viral vectors
77
Why is somatic cell gene therapy difficult to do? (3)
- Vector with healthy alleles needs to be inserted into affected cells - Engineered plasmids need to become incorporated into the nucleus - Gene expression of healthy allele needs to start and be maintained
78
Explain the key points of somatic cell gene therapy (6)
- Cures disease in the individual - Reduces symptoms - Cannot be inherited/passed to offspring - Only some cells have functional gene - Healthy allele passed on in mitosis - Repeated treatments needed due to cell deaths
79
How is the CFTR gene delivered?
Gene added to a plasmid vector Wrapped in liposome so it can pass through cell membrane
80
What is germ line cell gene therapy?
Copy of a fucntional gene put into perm, egg or embryo cells, so all cells have the fucntioning gene - Only used in animals + plants; Banned for human use
81
What are the advantages of germ line cell gene therapy?
- Functioning healthy gene can be inherited/passed to offspring - Permanent so doesn't need repeat treatment
82
Define bioinformatics
The develpoment of software and computing tools to organise and analyse raw biological data
83
Define computational biology
Use of data to build theoretical models of biological systems
84
List 3 uses of computational biology
- Working out the 3D structure of proteins - Identifying genes linked to specific diseases - Identifying evolutionary relationships between organisms
85
What does sequencing the genome of pathogens enable? (4)
- Doctors to find the source of an infection - Doctors to identify antibiotic resistant bacteria - Scientists to track the progress of an outbreak of a diseases - Scientists to identify regions of the genome of pathogens that new druygs could act on for vaccines
86
Define DNA barcoding?
Using particular sections of the genome common to all species but vary between them to identify specie
87
Define proteomics
The study of amino acid sequencing of an organism's entire protein complement
88
Define synthetic biology
The ability to sequence the genome of organisms and understand how eah sequence is translated into amino acids
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