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Biology A2 > Manipulating Genomes, 6.3 > Flashcards

Manipulating Genomes, 6.3 Flashcards

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1
Q

What is gene sequencing?

A

Allows genes to be isolated and can read the base sequence of a length of DNA

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2
Q

What is gene sequencing based off?

A

A technique called Sanger Sequencing.

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3
Q

What is the first stage of Sanger sequencing?

A

Add to each one of the four test tubes (A,T,C and G)

  1. Sample of DNA to be sequenced
  2. A radioactive primer
  3. Four DNA nucleotides
  4. DNA polymerase
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4
Q

Describe the stages of Sanger sequencing after the four test tubes have been prepared?

A
  1. Add a small amount of a special modified dideoxy
    nucleotide that cannot for a phosphodiester bond and
    so stops further synthesis of DNA
  2. From time to time the modified base will be added to
    the growing chain and synthesis will stop
  3. A full range of DNA will be synthesised ranging from
    short to full length and will stop at the specific base
  4. Contents of the four tubes are run side by side in
    electrophoresis
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5
Q

What are the advantages and disadvantages of the Sanger sequencing method?

A

Efficient and safe. Time consuming and very costly.

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6
Q

How do we clone DNA?

A
  1. Gene to be sequenced was isolated, using restriction enzymes
  2. DNA then inserted in a bacterial plasmid which divides and produces many copies of the DNA
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7
Q

What method of gene sequencing do we use now?

A

High throughput sequencing

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8
Q

What are the different stages of pyrosequencing?

A
  1. DNA cut into fragments, 300-500 base pairs
  2. Fragments are degraded to become single stranded
  3. Sequencing primer is added and DNA is incubated
    with DNA polymerase, ATP sulfurylase, luciferase,
    apyrase, APS and lucifern
  4. One of ATP, TTP, CTP, GTP is added and they are
    incorporated into a complementary strand
  5. When they are incorporated two extra phosphoryls are release as pyrophosphate - dephosphorylate
  6. In the presence of APS the enzyme ATP sulfurylase converts the pyrophosphate to ATP
  7. In the presence of ATP the enzyme luciferase converts luciferin to oxyluciferin. This generates light which can be detected by a camera.
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9
Q

How does the light enable us to detect a DNA sequence in pyrosequencing?

A

The amount of light produced is proportional to the amount of ATP. Indicates how many of the same type of activated nucleotides were incorporated together

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10
Q

How many reads can occur during pyrosequencing?

A

One million reads occur simultaneously. 10hour run generates 400million bases of sequencing information

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11
Q

What is meant by bioinformatics?

A

Stores the huge amounts of data generated. Would have been impossible to store and analyse data prior to computers and microchips.

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12
Q

What is the human genome project?

A

Sequenced in 2003. Contained around 24,000 genes - not much more than in a mouse.

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13
Q

Who do we share 99% of our DNA with?

A

Chimpanzees

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14
Q

What has a change in the gene FOXP2 caused in humans?

A

Speech

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15
Q

How different is the human genome from other organisms genomes?

A

Not very different. Highly conserved. Genes have been slightly alter in different organisms but no completely different genes

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16
Q

How can comparing organisms be biologically useful?

A
  1. Determine evolutionary relationships

2. Lead to organisms being reclassified

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17
Q

How can we compare different organisms using gene sequencing?

A

Get DNA from bones or teeth of extinct animals that can be amplified and sequenced to look the animals

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18
Q

How similar is DNA between individuals eg humans?

A
  • All humans are genetically similar.
  • About 0.1% of our gene is not shared with others
  • Differences arise from mutations
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19
Q

What effect does methylation of chemical groups have?

A

Plays a major role in regulation of gene expression. Mapping the methylation can help researchers to understand the development of certain diseases.
- eg certain types of cancer

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20
Q

How can an amino acid sequence be determined?

A

If the organisms gene is sequenced and they know which gene codes for a specific protein they can determine the proteins of the primary structure

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21
Q

What is synthetic biology?

A

Designing and building useful biological devices and systems

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22
Q

What are some examples of Synthetic biology?

A

Biofuels. Biomedicine. Food production. Chemicals. Biomaterials. Biosensors.

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23
Q

What is the basic principle of genetic engineering?

A

Remove a gene from one organism and transfer into another so the gene is expressed in a new host

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24
Q

What are the three main things that must be done in genetic engineering?

A

Obtain the wanted gene. Clone the gene to produce many copies. Insert a copy of the gene into host DNA.

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25
Q

How is DNA obtained from the amino acid sequence?

A

If amino acid sequence is know then the DNA code can be worked out and made in a lab.

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26
Q

How is DNA obtained from isolating the mRNA?

A
  1. Isolate the mRNA from cells whcih express a lot of the desired protein
  2. A complementary strand to the mRNA is made using reverse transcriptase
  3. The hybrid mRNA/DNA is separated.
  4. DNA polymerase is added to make double stranded DNA using cDNA as a template
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27
Q

How is DNA obtained from the entire orgnasims genome?

A
  1. DNA is cut using restriction endonucleases - means there is a high chance of a fragment without the right gene
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28
Q

Describe a restriction endonuclease enzyme

A

They are palendromic. Result in blunt or sticky ends. Cut off at specific bases.

29
Q

Why do we want sticky ends over blunt ends?

A

Sticky ends have free bases for more nucleotides to attach to

30
Q

What is a vector?

A

How the length of DNA is inserted into the host. eg baterial plasmid

31
Q

How is the DNA inserted into the plasmid?

A

Plasmid is cut with same restriction enzymes to make sticky ends. DNA can join into the loop of plasmid.

32
Q

Aside from DNA joining into the loop of plasmid what else could happen?

A

DNA forms a circle. Plasmid joins with unwanted gene. Plasmid joins back up again

33
Q

How is the plasmid taken up into the bacteria?

A
  • Bacteria needs to be in the presence of calcium ions to take up the plasmids.
  • Plasmid is added to bacteria grown in the presence of calcium chloride solution
  • About 1% pf bacteria take up some DNA
34
Q

What two genes are inserted into the loop of plasmid?

A

Ampicillin resistant gene and tetracycline resistance gene

35
Q

How are bacteria that have taken up the plasmid identified?

A
  • Bacteria transferred to a medium containing ampicillin
  • Only those containing the plasmid will grow
  • Know which have the plasmid but not the recombinant
36
Q

How do we get rid of plasmid without the wanted gene?

A
  • Replica plating
  • Plasmids tetracycline resistance gene is disrupted when a gene is inserted, it won’t work
  • Use absorbant cloth and get bacteria from each colony
  • Transger the bacteria onto tracycline plate
  • Non-recombinant bacteria will grow on the plate
37
Q

How do we identify the correct recombinant bacteria?

A
  • Us a gene probe
  • Labelled with radioactive nucleotides
  • Probe will bind to the plasmid and its presence is found through radioactivity
  • Could also make the gene resistant to an antibiotic
38
Q

What is a gene probe?

A

Short single stranded length of DNA. Complementary to a section of DNA being investigated

39
Q

What is the Polymerase Chain Reaction?

A

Technology than can amplify a short length of DNA to thousands of millions of copies

40
Q

Why is the Polymerase Chain Reaction useful?

A
  • Amplify one sample to create a large enough sample for analysis
  • Can create a DNA profile
41
Q

What does the Polymerase Chain Reaction rely on?

A

DNA has 2 anti-parallel strands. Each strand has a 5’ end and a 3’ end. Base pairing is complementary.

42
Q

What is needed for the Polymerase Chain Reaction?

A
  • DNA fragments to be copied
  • DNA polymerase, need an enzyme tolerant to heat, taken froom bacteria in hot springs
  • Primers, short pieces of DNA that bind to the start of each end of fragment
  • Free nucleotides
  • Thermocycler, a machine that varies the temperature
43
Q

Describe the process of denaturing in the Polymerase Chain Reaction

A
  • Denature means to separate the strands into 2 separate strands of DNA
  • Heat the DNA sample up to 96degrees
  • This breaks the hydrogen bonds
44
Q

Describe the process of annealing in the Polymerase Chain Reaction

A
  • Anneal means to add
  • 2 primers are added to the separated DNA strands
  • Temperature is cooled to 65degrees so the primers bind
  • The attachment of primers signals polymerase to start synthesising
45
Q

Describe the process of extension of DNA in the Polymerase Chain Reaction.

A
  • 2 polymerase molecules attach to the 2 primers on the 2 DNA strands
  • Polymerase moves along the strand
  • New complementary DNA is created
  • Temperature rises to 72degrees
46
Q

How quickly does the DNA increase?

A
  • Increases exponentially

- Do this process as many times as needed to create millions of strands

47
Q

How are strands identified after the Polymerase Chain Reaction?

A

Electrophoresis - separates them according to size.

48
Q

What are some applications of the Polymerase Chain Reaction?

A

Tissue typing - reduce risk of rejection for transplants. Detecting mutations - parents tested, parental screenings. Forensic science - amplified for DNA testing. Paternity test. Detecting mutations.

49
Q

What is electrophoresis?

A

Used to separate fragments of DNA according to their size.

50
Q

Describe the process of electrophoresis.

A
  1. Tank set up containing agarose gel
  2. Fragments added to wells in the gel - need coloured marker so yo ucan see them
  3. A current is passed through the gel
  4. DNA has a phosphate backbone so overall it as a negative charge - moves towards the anode
  5. Small fragments move fast and therefore will move further than larger fragments
51
Q

What are DNA arrays?

A
  • Ordered arrangement
  • Arrays of DNA probe used to detect the presence of specific sequences in the samples of DNA
  • Used in the detection of cystic fibrosis
52
Q

What do we use for Polymerase Chain Reaction?

A

Short Tandem Repeats - common and non coding

53
Q

What is the process of DNA profiling?

A
  1. DNA obtained eg mouth swab, blood, hair
  2. DNA digested with restriction enzymes
  3. Fragments separated by gel electrophoresis and stain
  4. A banding pattern can be seen
  5. Banding is compared to other individuals DNA
    (Related individuals will have a more similar banding pattern)
54
Q

What is gene therapy?

A

Treatment of genetic diseases by providing the sufferer with a corrected copy of the defective gene

55
Q

What is meant by transfection?

A

Inserting the corrected gene into the cell

56
Q

Describe Somatic Cell Therapy

A
  1. Copies of the corrected gene are inserted into the body cells of the sufferer
  2. Does not prevent the disease from occurring in the next generation
  3. Has to be repeated lots of times as the effects do not last long
57
Q

Describe Germ Line Therapy

A
  1. Corrected gene is inserted into a fertilised egg produced via IVF
  2. Cells of the embryo will contain the corrected gene when cells divide by mitosis
  3. Germ cell therapy is permanent and also ensures offspring inherit corrected gene
  4. Currently illegal
58
Q

What causes Cysytic Fibrosis?

A

CFTR gene - deletion mutation of 3 bases. CFTR protein can no longer be produced

59
Q

What is the role of the CFTR gene?

A

Transport ions across epithelial cell membranes. Water then follows by osmosis - membranes kept moist and mucus runny

60
Q

What are the effects of Cystic Fibrosis?

A

Thick, sticky mucus. Accumulate sin the lungs causing breathing difficulties. Mucus block pancreas so release of digestive enzymes effected. Sperm duct blocked/

61
Q

What are two vectors used to introduce corrected genes?

A

Harmless virus. Liposomes

62
Q

How are liposomes used as vectors?

A
  1. Normal gene isolated and inserted into plasmids
  2. Recombinants plasmids placed in bacteria
  3. Gene markers pick up cells with the plasmids
  4. Bacteria cloned
  5. Plasmids extracted and wrapped in lipid molecules - forms liposomes
  6. Liposomes sprayed into the patients ariways
  7. Liposomes pass easily through the plasma membrane of cells and into the nucleus
63
Q

How are viruses used as vectors?

A
  1. Modify adenoviruses genes involved in replication to make them harmless
  2. Grow adenoviruses in epithelial cells in a lab. Add recombinant plasmids that contian functional genes
  3. Recombinant plasmid taken up by adenovirus. Gene becomes part of virus DNA.
  4. Virus isolated from epithelial cells and purified
  5. Virus sprayed into the nostrils of patients via aerosol
64
Q

State some of the problems with Gene Therapy

A
  • New technology
  • Limited success in trials
  • Liposomes may not be small enough to pass into cells
  • Poor expression of the CFTR genes
  • Adenoviruses may cause infection
  • Adenoviruses may trigger immune response or patients will develop immunity
65
Q

What has gene therapy been used to treat?

A

SCID - a mutation of the ADA gene, normal ADA destroys toxins that will kill white blood cells.

66
Q

What are some examples of selective breeding?

A

IVF. Artificial insemination. Cloning. Embryo translation. Genetic engineering

67
Q

What are the impacts of GM?

A
  1. Would help with the problem

2. Reduce genetic diversity, suffer from disease, expensive, unknown effects

68
Q

What are the impacts of Gene therapy? (Somatic gene therapy)

A
  1. Cure symptoms, extend lifespan reduce suffering

2. Virus may cause disease, invasive procedure, temporary, rejection, animal testing concerns

Biology A2 (13 decks)

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