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BIO > Manipulating Genomes > Flashcards

Manipulating Genomes Flashcards

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Biology 101 flashcards
1
Q

What is DNA sequencing?

A

Determining the precise order of the nucleotides in an entire genome.

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2
Q

What can be linked to the increased ability to sequence genomes?

A

Development of high-throughput sequencing (pyrosequencing/massively parallel sequencing)

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3
Q

What are the names of the old and new DNA sequencing methods?

A

Old: SANGER method (chain termination method)
New: High-throughput sequencing (pyrosequencing).

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4
Q

What is the initial step of both DNA sequencing methods?

A

Chromosomes are digested using restriction enzymes to produce fragments short enough to be sequenced.

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5
Q

How are fragments amplified in the SANGER method compared to the new method?

A

Old: bacteria used
New: PCR used

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6
Q

In which DNA sequencing method is gel electrophoresis used?

A

The SANGER method

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7
Q

In which DNA sequencing method are fluorescently labelled terminator nucleotides used?

A

The SANGER method

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8
Q

In which DNA sequencing method are microbeads and an array of wells used?

A

High-throughput sequencing

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9
Q

What is the final step of both DNA sequencing methods?

A

Once the sequence of each DNA fragment has been determined, they can be stitched together using computer software as the fragments are all overlapping.

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10
Q

What is the main benefit of high-throughput (pyro) sequencing?

A

It is much faster as around a million fragments can be read at the same time in the array of wells.

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11
Q

What is bioinformatics?

A

The development and use of computer software to analyse large data sets, such as from high-throughput sequencing.

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12
Q

What is computational biology?

A

The use of computers to build theoretical models of biological systems, in order to predict molecule interactions.

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13
Q

What is genomics?

A

The use of DNA sequencing and computational biology to analyse the structure and function of genomes.

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14
Q

What is proteomics?

A

Determining the amino acid sequences of the different proteins made by an organism.

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15
Q

What are 3 applications of comparative gene mapping?

A

Identify the genetic components of diseases.
Sequencing the genomes of pathogens.
Determining evolutionary relationships (phylogeny)

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16
Q

What is the use of sequencing the genomes of pathogens?

A

Identify new targets for drugs and vaccines.

Assess antibiotic resistance to advise the suitable use of antibiotics.

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17
Q

What is the use of identifying the genetic component of diseases?

A

Helps develop more effective treatment.

Helps identify genetic factors early.

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18
Q

How can phylogeny be determined using comparative gene mapping?

A

Looking at genes which are highly conserved within species.

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19
Q

What part of the genome is used for genetic profiling? Why?

A

The length of non-coding regions (introns or microsatellite regions) as there is much intraspecific variation in the lengths of these regions.

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20
Q

How is the banding pattern of DNA visualised after gel electrophoresis (in genetic profiling)?

A

The DNA is transferred to a nitrocellulose filter (southern blotting), the DNA is denatured, and then hybridized with DNA probes.

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21
Q

What is hybridization?

A

When a DNA probe anneals with its complementary base sequence.

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22
Q

How are DNA probes used in genetic profiling?

A

The DNA must first be denatured after southern blotting, so that the hybridization of the DNA probes can occur.
Radiolabelled DNA probes absorb X-rays.
Fluorescent tags emit light when shone with UV light.

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23
Q

How is genetic profiling (and specific DNA probes) used in pharmacogenomics?

A

To predict the response of an individual to therapeutic drugs.
Designing personalised treatments for cancer patients.

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24
Q

How else is genetic profiling used other than in pharmacogenomics and pre-implantation genetic diagnosis?

A

Finding good genetic matches for organ/marrow donation.
Forensics.
Paternity testing.

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25
Q

What is pre-implantation genetic diagnosis?

A

The use of IVF and genetic profiling (and DNA probes) to screen for serious genetic diseases.

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26
Q

Which couples are allowed to use pre-implantation genetic diagnosis?

A

Couples with a family history of genetic disease.

The list of diseases for genetic screening is strictly regulated by ethics committees.

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27
Q

What is DNA amplification?

A

Using PCR, lots of DNA can be made that is genetically identical to the original sample.

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28
Q

What are the 3 stages of PCR? What temperatures do they run at?

A

Denaturation 95
Primer Annealing 55
Extension 72

29
Q

What happens in denaturation in PCR?

A

The temperature is raised to 95 degrees, causing the strands of the DNA double helix to separate, breaking the weak hydrogen bonds between complementary bases.

30
Q

What happens in primer annealing in PCR?

A

The temperature is decreased to 55 degrees, allowing DNA primers to anneal to complementary nucleotide sequences.

31
Q

What happens in extension in PCR?

A

The enzyme taq polymerase synthesises complementary strands of DNA from free nucleotides, starting at the primers.
72 degrees is the optimum temperature for this process.

32
Q

Why is the PCR tube thin-walled?

A

To allow temperature changes in the thermocycler.

33
Q

What is the PCR mix?

A 
Sample DNA
Forward and reverse primers
Taq polymerase
Free DNA nucleotides
MgCL2 (taq polymerase cofactor)
34
Q

What is taq polymerase?

A 
A thermostable (doesn't denature at 95) form of DNA polymerase.
Originates from the thermophilic bacterium thermus aquaticus.
35
Q

What is the role of forward and reverse primers in PCR?

A

Primers only bind at the target locus on the gene as they are complementary to the DNA strand.
Forward and reverse primers locate the sequence of DNA to be amplified and allow DNA polymerase to begin adding nucleotides to the 3prime end.

36
Q

What is the role of a thermocycler machine in PCR?

A

Quickly changes the temperature, performing many cycles of PCR automatically. After only 25 cycles, millions of identical DNA strands have been produced.
The components of the PCR mix are very expensive.

37
Q

What is electrophoresis?

A

A type of chromatography that causes charged molecules to move through a gel medium (agarose gel) under the influence of an electric current.

38
Q

How does DNA travel in gel electrophoresis?

A

DNA is negatively charged due to the phosphate group in its backbone, so DNA travels towards the anode.

39
Q

Why do larger DNA fragments travel more slowly in gel electrophoresis?

A

They experience a greater retarding force as smaller fragments travel through pores in the agarose gel more easily.

40
Q

What is the purpose of gel electrophoresis?

A

To fractionate a mixture of DNA fragments of different sizes according to their length.

41
Q

What are restriction endonucleases?

A

Enzymes which cut the DNA molecule at a specific palindromic recognition sequence, their active sites are complementary to their specific recognition sequence.

42
Q

What is the result of digestion by a restriction enzyme?

A

The DNA is cut via a hydrolysis reaction, leaving the two fragments with blunt or sticky ends.

43
Q

What are sticky ends and why are they useful?

A

Small tails of unpaired bases left after digestion by a restriction enzyme.
Sticky ends can be used to anneal DNA fragments.

44
Q

What enzymes are used in genetic engineering?

A

Restriction enzymes
DNA ligase
Reverse transcriptase
Taq polymerase

45
Q

What is the use of DNA ligase in genetic engineering?

A

Used to join together sticky ends, reforming the sugar-phosphate backbone, forming a phosphodiester bond.

46
Q

What is the role of reverse transcriptase?

A

Uses mRNA molecules as a template to form cDNA

47
Q

What is cDNA?

A

Copy DNA, contains no introns, only coding regions so can be inserted into a plasmid. (bacteria have no introns)

48
Q

What are transformed organisms/cells?

A

Organisms/cells that have had their DNA altered by genetic engineering.

49
Q

What is recombinant DNA?

A

A molecule of DNA that consists of material from two sources that has been joined together.

50
Q

Why are bacteria or yeast good DNA hosts?

A

Grow quickly
Easily manipulated
Contains plasmids (naturally occurring gene vectors)

51
Q

What are the 4 steps in genetic engineering of bacteria?

A

Isolate the gene to be cloned
Produce recombinant plasmid
Transform bacteria
Select transformed bacteria

52
Q

How is the gene to be cloned isolated in gene cloning?

A

mRNA is extracted from the cytoplasm of a cell that expresses the gene.
Reverse transcriptase is used to produce cDNA.
PCR is used to amplify the cDNA.

53
Q

How are recombinant plasmids produced?

A

The gene and plasmid are cut with identical restriction enzymes to obtain complementary sticky ends, DNA ligase is then used to join the gene and plasmid, forming the recombinant plasmid.

54
Q

How is the bacteria transformed?

A

Plasmid uptake by the bacterial cells.

The transformed cells are selected.

55
Q

How can you encourage plasmid uptake?

A 
Electroporation
Viruses
Microinjection
Ti plasmids
Liposomes
56
Q

What is electroporation?

A

Cells are subjected to a high-voltage pulse which temporarily disrupts the membrane and allows the vector (plasmid) to enter the cell.

57
Q

How are transformed bacterial cells selected?

A

Plasmids all contain an antibiotic resistance marker gene, used to determine which bacteria have taken up the plasmids.
Plasmids all contain a fluorescence marker gene, which is disrupted if the plasmid becomes transformed by the target gene.
Bacteria that survive the antibiotic and do not fluoresce have taken up the transformed plasmid.

58
Q

What is gene therapy?

A

Introduction of genes into an individual with abnormal genes, so allowing the individual to produce proteins they would otherwise be unable to, such as insulin in type 1 diabetes.

59
Q

What is involved in cell-based gene therapy?

A

Stem cells taken from the patient, cells have genes added (transfection), transformed cells are administered to the patient.

60
Q

What is the role of vectors such as viruses in gene therapy?

A

Used to insert genes into the patient’s cells.
Different vectors used for genes of different sizes.
Some vectors integrate genes into chromosomal DNA, offering life-long protection.

61
Q

What is somatic gene therapy?

A

Targets cells which are not gametes. Two methods:
Addition of genes (augmentation) (e.g: treat cystic fibrosis)
Killing of specific cells (e.g: treat cancer)

62
Q

What is pharming?

A

Genetically modifying animals to produce human proteins (as bacteria cannot produce some complex proteins). Or to produce organs for transplant.
or
Creating animal models for the development of new treatments.

63
Q

What is a positive/negative of pharming?

A

Can lead to the production of new/less expensive pharmacology.

Potential danger to ecosystems if modified organisms spread.
Treats animals as solely a human commodity

64
Q

What are positives/negatives of introducing insect resistance/weed-killer resistance to GM soya?

A

Higher yield.
Easier+less expensive to grow.
Reduces the use of pesicides => better for ecosystem.

Weeds may become weed-resistant/ insect resistant.
GM crop seeds are more expensive and must be bought each year. (dependence on large companies).
Insects may become resistant to insecticides.

65
Q

What are some concerns over the use of GM pathogens for research into treatment of diseases?

A

Potential pathogen outbreak.
Potential use of GM pathogens for bio-warfare.
Concern about administration of pathogens as treatment.

66
Q

What are positives/negatives of patenting of genetically modified organisms/GM technology?

A

Patent owners can profit from their research, funding new research.
Allows competition between scientists.

Poorer farmers cannot afford GM crops.

67
Q

What kind of gel is used in electrophoresis?

A

Agarose gel

68
Q

Which antibiotic is most often used when selecting transformed bacterial cells?

A

Ampicillin

69
Q

What is the significance of Ti plasmids?

A

Ti plasmids are the only plasmid taken up by plant cells.

Ti plasmids come from the bacterium agrobacterium tumerfaciens.

BIO (33 decks)

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