Biology > Manipulating Genomes > Flashcards

Manipulating Genomes Flashcards

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1
Q

What are the 2 main approaches to using gene therapy for treating or preventing genetic disorders

A

Counteracting recessive disorders
Silencing faulty dominant alleles

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2
Q

How can gene therapy treat genetic disorders

A

Identify the abnormal gene responsible for the disorder
Engineer normal, functional versions of this gene by removing it from healthy cells or synthesis in lab
Deliver normal allele to nuclei of target cells using vector

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3
Q

What is somatic gene therapy

A

Replaces mutant alleles with healthy alleles in affected body cells to treat diseases

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4
Q

What are the target cells of SCGT

A

Somatic (body cells)

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5
Q

Are gene modifications inherited by offspring in SCGT

A

No

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6
Q

What is germline gene therapy

A

Involves inserting healthy allele into embryo to prevent genetic diseases from birth

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7
Q

What are target cells for germline therapy

A

Germ cells (egg/sperm)
Embryos

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8
Q

Are modifications inherited in germline therapy

A

Yes

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9
Q

What are the general ethical issues of both gene therapies

A

Potential misuse for enhancing aesthetic attributes rather than medical needs
Risk of causing unintended harm
Diverts scarce healthcare resources
High in cost - restricts access

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10
Q

What are the ethical benefits of gene therapy

A

Extends lives by treating diseases
Enhances quality of life
Germline allows carrier parents to have children free from genetic disorders

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11
Q

What are the ethical issues with SCTC

A

Delivering healthy alleles to cells is challenging
Getting healthy alleles into the nucleus is challenging
Maintaining the expression of healthy alleles is challenging
Effects are short term as semantic cells have limited lifespan and are replaced by cells with the faulty allele

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12
Q

Ethical issues with germline therapy

A

Rights of unborn child are violated - cannot provide consent
Causes irreversible changes - long term outcomes not fully understood
Could be used to select desirable traits

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13
Q

Advantages of GM crops

A

Involves less labour than traditional herbicides and pesticides
Cheaper than these
Herbicide resistant allow use of herbicides to kill weeds without harming crop - ^ yield
Insect resistant less likely to be killed by pests - ^ yield
Insect resistant reduce need for pesticides, benefiting environment

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14
Q

Disadvantages of GM crops

A

Encourages monocultures - reducing biodiversity
Herbicide resistant may cross breed with wild plants, creating herbicide resistant weeds
Loss of traditional crop varieties - reducing genetic diversity - makes vulnerable to disease & climate changes
Non target insects could be harmed by toxin coded for by insect resistant crops

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15
Q

Advantages of GM farmed animals

A

Improves quality (enhance disease resistance)
Improves quantity (enables year round reproduction)
Improves productivity (faster growth)

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16
Q

What is pharming

A

Involves production of pharmaceuticals & human medicines by inserting human genes into other animals, allowing for extraction of pharmaceutical proteins from GM animals’ milk/blood at ^ yields

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17
Q

Advantages of pharming

A

Enables mass production of rare treatments
Makes drugs more accessible

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18
Q

Disadvantages of pharming

A

Raises animal welfare concerns
Can lead to animals being viewed solely as commodities

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19
Q

Advantages of GM pathogens

A

Offers potential treatments for diseases prev deemed incurable
Can produce essential medicines, antibiotics and enzymes eg insulin
Useful in creating vaccines
Facilitates creation of DNA libraries for reseach

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20
Q

Disadvantages of GM pathogens

A

Carries risk of accidental infections and disease outbreaks
Danger that engineered pathogens could revert to original harmful form
Could be misused in biological warfare

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21
Q

What are the ethical concerns with patenting GM organisms

A

Legal patenting raises questions about global access
Affects smallholder farmers in developing countries who may be unable to afford patented seeds due to high costs

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22
Q

What is genetic engineering

A

Deliberate manipulation of genetic material to modify an organisms’ characteristics

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23
Q

What is recombinant DNA

A

DNA altered to contain nucleotides from 2 different organisms
Allows DNA fragments to be transferred between organisms

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24
Q

What are the key stages of gene transfer

A
  1. Desired gene identified and isolated
  2. PCR
  3. Gene inserted into vector
  4. Vector delivers gene into cells into different organism
  5. Cells with new gene identifies using marker genes
  6. Cells with new gene cloned
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25
What are transgenic organisms and why are they possible
Organisms which can express transferred gene to produce encoded protein and express new genetic traits Due to universal nature of genetic code & similarity of transcription and translation across organisms
26
Main methods of producing DNA fragments
1. Making complementary DNA (cDNA) using reverse transcriptase 2. Cleaving DNA from donor organism using restriction enzymes
27
How does reverse transcriptase produce DNA fragments
1. Extract mRNA from cells 2. mRNA reverse transcribed using reverse transcriptase 3. CDNA is formed and isolated from mRNA strand 4. free nucleotides, cDNA, DNA polymerase form other strand of DNA, reforming desired gene
28
Why is mRNA used when using reverse transcriptase to produce DNA fragments
Easier to extract than DNA
29
What do restriction enzymes do
Recognise & cut DNA at specific palindromic nucleotide sequences to isolate gene fragments
30
What are palindromic sequences
Sections of DNA where the nucleotides sequence reads the same in opposite direction on antiparallel strands
31
How do restriction enzymes cut DNA
1. DNA incubated with restriction enzymes 2. Restriction enzymes identify palindromic sequences in DNA double helix and cut double stranded DNA if recognition sequence present 3. Recognition sequences at either end of desired DNA fragment allow restriction enzymes to separate fragment from rest of DNA to obtain desired gene 4. Enzymes cut target gene fragment out via hydrolysis 5. Different restriction enzymes cut at different sequences based on active site shape
32
What are sticky ends
Short overhanging sequences of unpaired bases that can bind to other DNA fragments
33
How is DNA inserted into vectors
1. Vector cut open at specific site using restriction enzymes creating sticky ends 2. Same restriction enzyme used to cut target DNA fragment, creating complementary sticky ends 3. DNA ligaments forms phosphodiester bonds between sugar and phosphate groups on two strands of DNA, joining sticky ends of vector and DNA fragment together 4. Newly formed molecule = recombinant DNA
34
What could vectors which transfer DNA be
Plasmids or bacteriophages
35
What are plasmid vectors
Small circular DNA molecules
36
What are bacteriophage vectors
Viruses that infect bacteria
37
How do marker genes indicate which hoste cells took up recombinant DNA
1. Inserted into vectors alongside target genes 2. Transformed cells are cultivated on selective agar plates 3. Only transformed cells display characteristics encoded by marker genes 4. These transformed cells can then be cultured to mass produce target DNA fragment through cellular replication
38
Features of VNTRs
Present across genomes of most eukaryotes Extensive variability in sequence and length Length and location are inheritable
39
Main steps of DNA profiling
1. Extraction - (&lification with PCR) 2. Digestion - restriction enzymes used to cut DNA into fragments at points near VNTR sequences 3. Fragment separation - gel electrophoresis & denaturation to single strands 4. Hybridisation - specific radioactive/fluorescent probes bind to complementary VNTR sequences 5. Development - positions of probes revealed, creating barcode like pattern of DNA bands
40
Key applications of DNA profiling
Establishing paternity Identifying suspects from crime scene DNA Supporting criminal convictions Identifying risk of genetic disorders Selecting desirable traits in plants/ animal for selective breeeding while preventing severe inbreeding
41
Limitations of DNA profiling
Environmental contamination may compromise results Close genetic relatives could have similar fingerprints
42
What is PCR
Method of amplifying DNA
43
Components required for PCR
DNA fragment Primers DNA polymerase (Taq) Free nucleotides Themocycler
44
Why is taq polymerase used
Can withstand high temps without denaturing to remain active throughout process
45
What are the 3 main stages of PCR
1. Separation (denaturation) - heating DNA separates H bonds between complementary strands 2. Addition of primers (annealing) - primers attach to specific starting pints on each o separated DNA strands by forming H bonds 3. DNA synthesis (extension) - taq polymerase adds free nucleotides to the ends of the primers, extending DNA strand to form complete copy
46
What temps are each of the PCR steps at
Denaturation - 95 Annealing - 55 Extension - 72
47
Advantages of PCR
Rapid speed Precision (automated nature of thermocycler) Low DNA needs No cells needed
48
What is gel electrophoresis
Technique used to separate molecules eg DNA based on size using electric current applied to agarose gel matrix
49
How to set up gel electrophoresis
1. Insert gel tray with solidified agarose gel into gel tank 2. Ensure wells are close to negative electrode 3. Pour buffer solution over gel until submerged to maintain constant pH
50
How to load samples in gel electrophoresis
1. Mix DNA samples with loading dye 2. Deposit equal vols of each sample into wells with micropipette 3. Touch micropipete tip to buffer, not bottom of gel to prevent damaging gel
51
What happens during electrophoresis
1. Voltage applied across gel 2. Fragments of DNA move toward anode 3. Small fragments travel faster & thus separate by size 4. Continue process until dye approaches end/ 30 mins
52
Why does the size based separation occur in electrophoresis
Gel’s mesh matrix slows down larger fragments more
53
What techniques have improved since the original Sanger sequencing
Substitution of radioactive labels with fluorescent tags for safety and efficiency Enhancements in scaling up & automation to process more samples at once Intro of capillary sequencing
54
Outline process of DNA sequencing
1. DNA mixed with primers, DNA polymerase, normal nucleotide bases and ‘terminator’ bases 2. Split into single strands & copied multiple times 3. DNA polymerase adds nucleotides to single stranded template to start rebuilding new DNA strands 4. Terminator base added, DNA synthesis stops, each tagged with unique fluorescent colour 5. Produces DNA fragments of all lengths 6. DNA fragments separated by length (electrophoresis) 7. Laser then detects fluorescent colours of terminator bases 8. Computer software analyses fragments to reconstruct sequence
55
What have new generation sequencing technologies facilitated
Massively parallel sequencing Exponentially increased speed Reduced costs
56
What is massively parallel sequencing
Simultaneous sequencing of millions of DNA fragments
57
What is bioinformatics
Involves developing software, computing tools, math models to collect, store, and analyse biological datasets
58
What is computational biology
Field uses bioinformatics tools & biological data to model DNA systems and processes
59
What are genomics
Applies dna sequencing and computational biology to study genomes of organisms
60
What are the benefits of sequencing pathogen genomes
Identifying the sources and transmission routes of diseases Detecting antibiotic resistant strains Developing new treatments and vaccines by identifying drug targets Monitoring disease outbreaks
61
What are the advantages of dna barcoding
Fast and affordable sequencing Classification of new species Updating of classifications
62
What is genomics
Study of genomes using DNA sequencing and computational biology
63
What is proteomics
Examining the complete set of proteins produced by the genome
64
What is synthetic biology
Design and construction of new bio part, pathways, organisms
65
What are the potential applications of synthetic biology
Synthesising functional genes to replace faulty ones when treating genetic disorders Utilising microorganisms and bio systems to produce dugs Constructing fully artificial genomes
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