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Manipulating Genomes Flashcards

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Biology 101 flashcards
1
Q

What types of genes are used for DNA profiling, and why?

A

Introns because there will be some differences in base sequences in exons as they code for characteristics like eye colour

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2
Q

What is satellite DNA and the two types?

A
  • Within introns
  • Short, repeated DNA sequences
  • Always appear on the same area of chromosomes
    Minisatellite - 20 to 50 base pairs long, repeated 50-100 times (aka VNTR’s)
    Microsatellite - 2-4 base pairs, repeated 5-15 times (STR’s)
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3
Q

What is the basic/essential principle of DNA profiling?

A
  • Different people will have a different number of repeats
  • Therefore they will generate a different satellite pattern
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4
Q

What are the stages of DNA profiling?

A
  • Extract DNA and amplify it by putting it through PCR to replicate the same DNA
  • Digestion
  • Separation, carry out gel electrophoresis
  • Southern blotting carried out, where the DNA fragments are transferred onto a membrane
  • Hybridisation, add florescent dye and put under UV light, to visualise evidence
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5
Q

Explain the stage of digestion in DNA profiling

A

Add enzyme restriction endonucleases, which recognise specific DNA sequences (restriction sites) and cut up the DNA into smaller fragments whilst leaving satellites intact

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6
Q

Explain gel electrophoresis

A
  • Using electricity to separate DNA fragments
  • DNA fragments placed in wells on agar jelly on negative side of electrode, so negative DNA repels away and towards anode
  • Agar gel is immersed alkali to separate DNA double strands into single strands
  • DNA fragments separated by length, but can’t be seen until dye is added
  • Smaller fragments move through the gel faster
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7
Q

Briefly explain the 3 steps of PCR

A

Denaturation (95 degrees) - separating the double helix as H bonds are broken
Annealing (55) - primers bind to the start of the target DNA
Synthesis (72) - building up the new strand

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8
Q

What are primers?

A
  • Short DNA sequences that bind to the start of a gene you want to amplify
  • Designed to have complementary bases to gene
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9
Q

Explain the stage of synthesis in PCR

A
  • 72 degrees
  • Free nucleotides pair up to exposed bases by complementary base pairing
  • Taq polymerase joins up backbone of new strand to form phosphodiester bonds
  • Taq because human DNA polymerase will denature at 72 degrees
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10
Q

What is DNA sequencing and the 5 ingredients required?

A
  • Technique where we can map out the base sequence of an individual’s genome
    Need -
    1. DNA sample to be sequenced
    2. Free nucleotides (in excess)
    3. Coloured fluorescent labelled terminator bases
    4. DNA polymerase
    5. DNA primers
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11
Q

Explain the stages of DNA sequencing

A
  • Heat up to 95-96 degrees to denature DNA and separate the double helix and form 2 separate strands
  • Cool down to 50 degrees so the primers anneal to the single strands
  • Heat up to 60 degrees, DNA polymerase starts to join up paired nucleotides to synthesise new strand
  • Once terminator base binds, strand can no longer extend
  • Ends up with lots of strands of different lengths
  • Because free nucelotides are in excess, there will be less terminator bases than nucleotides, all possible DNA chains will be produced
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12
Q

Explain terminator bases

A
  • Have a hydrogen atom instead of a hydroxyl group at the 3 prime end of the ribose ring
  • Therefore they cannot form phosphodiester bonds with the next nucleotide
  • Sequence will end
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13
Q

What are the 2 ways we can read the data from DNA sequencing?

A
  • Do electrophoresis and southern blotting
  • Terminator bases are all labeled, so direct UV light onto membrane
    or
  • more recent way using laser over electrophoresis
  • generates pattern on computer
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14
Q

Define bioinformatics and computational biology

A

Bioinformatics - Development of software and computing tools needed to organise and analyse raw biological data, includes development of algorithms, mathematical models and statistical tests
Computational biology - uses data from bioinformatics to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances

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15
Q

What is synthetic biology?

A

The design and construction of novel biological pathways, organisms or devices, or the redesign of existing natural biological systems

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16
Q

What were the issues with using insulin from pigs?

A
  • Ensuring it was properly sterilised
  • Religion
17
Q

Outline the process of genetic engineering of insulin in bacteria

A
  • DNA extracted from human cell and desired gene identified using a process such as DNA sequencing
  • Plasmid extracted from bacteria as they contain a marker gene
  • Restriction endonuclease cuts out section where desired gene is, and same enzymes used to cut out marker gene from plasmid
  • Marker gene and desired gene bind on recombinant plasmid
  • Place recombinant plasmid in bacteria, which becomes transgenic as it carries a gene from a different type of organism
  • Bacterium cultured in agar plate
18
Q

What is a marker gene?

A

A gene used to determine if a nucleic acid sequence has been successfully inserted into an organisms DNA

19
Q

Explain the main method of isolating a gene in genetic engineering

A

Using restriction endonucleases - enzymes that recognise specific restriction site on genome, and cut it out
- They can either do one clean cut through the DNA sequence, or do a staggered cut
- Staggered cut produces sticky ends, which have exposed bases so can easily bind to plasmid

20
Q

What is the less common method of isolating a gene in genetic engineering?

A

Using reverse transcriptase
- Allow DNA to be first transcribed, leaving mRNA of desired gene
- Extract mRNA from cytoplasm, and mix with reverse transcriptase, to form complementary DNA (cDNA)
- Put cDNA into plasmid

21
Q

Explain how to insert the desired gene into the vector in genetic engineering

A
  • Extract plasmid from bacteria, or use a virus
  • Cut plasmid at the marker gene using the same restriction enzyme that was used to isolate the gene, to produce the same sticky ends
  • Blue marker gene produces blue pigment, but can’t be transcribed once desired gene is inserted into it as it’s cut in half, so can’t produce blue pigment
  • DNA ligase forms phosphodiester bonds between desired gene and plasmid/marker gene
  • Other marker gene, ampicillin resistant gene, so can survive in plate with ampicillin
  • Recombinant DNA formed
22
Q

What is the difference between DNA ligase and DNA polymerase

A
  • DNA ligase joins DNA fragments together at a single point
  • DNA polymerase joins individual DNA nucleotides together working across a whole template strand
23
Q

Explain the principle of transformation in genetic engineering

A
  • Recombinant plasmid needs to be placed into host cell (bacteria)
  • To make cell membrane more permeable, so plasmid more likely to be taken in
24
Q

Explain the three methods of transformation in genetic engineering

A

Method A
- Put plasmid and bacteria in calcium rich solution, and do a heat shock to create pores in membrane
Method B
- Electroporation, creates pores in membrane using electricity
- Small current passed through
Method C
- Electrofusion
- Have plasmid inside a vesicle and provide electric current through that and bacterial cell
- Causes them to assimilate and fuse together and plasmid passes through from vesicle to bacteria

25
Q

What are the 3 possibilities of bacteria at the end of genetic engineering, and what would happen when they’re all mass produced together?

A

1 - Bacteria didn’t take up plasmid, so bacteria remains the same
- Doesn’t have ampicillin resistant gene, so dies and won’t appear on plate
2 - Bacteria does take up plasmid, but plasmid was an original plasmid without desired gene
- Will grow and survive, and produce blue pigment
3 - Successful, bacteria took up plasmid, and plasmid was recombinant containing desired gene
- Will survive and grow, but won’t produce blue pigment

26
Q

What happens in mass production after genetic engineering?

A
  • Culturing of bacteria in agar gel with nutrients and ampicillin
27
Q

Explain the process of genetic engineering in plants

A
  • Uses agrobacterium tumefaciens, a bacterium that causes tumours in healthy plants
  • A desired gene, like pesticide production is placed in the Ti plasmid of A. tumefaciens, along with a marker gene like antibiotic resistance or flourescance
  • This is carried directly into the plant cells DNA
  • The transgenic plant cells form a callus, which is a mass of gentically modified plant cells, each of which can be grown into a new transgenic plant

Bio (19 decks)

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