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Genetics And Evolution > DNA Techniques > Flashcards

DNA Techniques Flashcards

1
Q

How does gel electrophoresis work

A

DNA migrates to the positive pole in electric field.
DNA charge is proportional to length so proportional to mass
Smaller molecules move faster through the gel = separation

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2
Q

What 2 fluorescent dyes are generally used with DNA

A

Intercalating agents - go between base pairs in double helix
Minor groove binding - molecules attach to grooves on outside

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3
Q

What colour light would you use to excite DNA binding dyes

A

Blue light

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4
Q

What colour light would you use to detect emission from DNA

A

Orange light

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5
Q

What makes the dyes fluorescent

A

One or more electrons can absorb light - causes excitation - jumps to a higher energy orbital and emits light as it falls

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6
Q

What is a polymerase chain reaction

A

Rapidly makes millions to billions of copies of a specific DNA sample, allows scientists to amplify small DNA to study it in detail

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7
Q

What factors had been discovered to make PCR possible

A
  • first DNA sequencing method had been developed
  • synthesis of oligonucleotides was possible
  • supply purified DNA polymerase to synthesis in vitro
  • several DNA polymerase were isolated
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8
Q

Describe the steps in the PCR cycle

A
  1. Mix DNA with nucleotide triphosphates and primers
  2. Increase temp to 92 to denature double helix
  3. Lower temp to 45-65 degrees for primers to anneal to add reverse complementary bases/sequence
  4. Polymerisation starts
  5. Increase temperature for optimum DNA polymerase
  6. DNA is duplicated from the RNA
  7. Denature and anneal again to keep creating copies
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9
Q

Advantages of the PCR

A

Sensitive - works well with little initial material
Specific - will exclusively copy the target sequence
Fast - each cycle takes 2 mins, 30 cycle ~2 hours

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10
Q

Disadvantages of the PCR

A

Knowledge limited - can’t apply to an unknown DNA molecule
Length limited - limited to smaller molecules
Hot - due to denaturing
Imprecise - limited to precision of polymerase alone

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11
Q

How do you copy RNA

A

Reverse transcriptase

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12
Q

What was the problem with trying to copy Viral RNA

A

Viruses don’t have a double helix DNA genome, just single stranded RNA.
Needed reverse complement sequence to copy genome template to make copies of genome.

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13
Q

What did scientists do to be able to copy RNA

A
  1. Purified a viral protein that can synthesis DNA using RNA as a template
  2. Incubated in vitro the purified viral genome and purified viral protein
  3. Added tritiated deoxytymidine triphosphate (dttp) - only used to make DNA
    Made tritium into a polymer - made of DNA
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14
Q

How is complementary DNA made

A
  1. Use oligo made of polytheoxytimidine - bind to polyA tails and synthesise reverse complement of RNA
  2. RNA degrades
  3. Single strand DNA used to synthesise can be used to synthesise the DNA strand that’ll be a copy of mRNA
  4. Double stranded DNA is made from mRNA
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15
Q

What is QRT-PCR

A

Quantitative reverse transcriptase - polymerase chain reaction.
Amplifies gene of interest from copy DNA

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16
Q

How does quantitative reverse transcriptase - PCR work

A
  1. Fluorescent dye is added to minor groove
  2. Measures the amount of double stranded DNA at the end of each extension step
  3. Done in a thermal cycler and spectrophotometer
  4. Plot a graph - show increase in double stranded DNA conc
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17
Q

How do you read a graph of QRT-PCR

A

The higher concentration of the target sequence = the earlier the curve goes up

18
Q

Aspects of short tandem repeats

A
  • simple short sequence repeats
  • 6nts
  • number of repeats is variable between individuals - valuable genetic markers
  • strand slippage = reason for variability
19
Q

How does strand slippage occur

A
  1. DNA polymerase synthesises repetitive DNA
  2. New strand detaches from template strand and is behind original position
  3. Results in copying one or more repeat twice = misalignment
  4. New strand is a different length to the template
20
Q

Give 2 forensic uses of microsatellites

A

High polymorphism

DNA fingerprinting

21
Q

For what reason might you want to build a genetic profile

A

Crime cases with ancestry.
Identifiying mass casualties after an explosion or natural disaster
Identify a species for food safety

22
Q

How do you visualise DNA with PCR

A
  1. Use primers flanking the microsatellite
  2. Run fragments in a gel
  3. Repeat with multiple microsatellites for richer band patterns
23
Q

Other uses of PCR

A

Genetic testing and data analysis
Ancient DNA
Creatures trapped in permafrost
Studies on evolution and geology

24
Q

What did scientists have to do to work out the function of 5’ UTR and 3’ UTR

A
  • isolate specific DNA segments
  • obtain the sequence
  • analyse the function
25
Q

What’s the difference between DNA cloning / molecular cloning / genetic engineering /recombinant DNA technology and genome editing

A

Genome editing takes place within an organism whereas everything else takes place in vitro, so in a test tube

26
Q

Aspects of bacterial plasmids

A
  • extra chromosomal DNA molecules
  • contain few genes
  • usually conferring resistance to antibiotics
  • circular
27
Q

States the 4 elements a plasmid needs

A
  • resistance genes
  • f factors
  • replication origin
  • internal structure
28
Q

What modification do plasmids need before they’re used for particle purposes

A
  1. Purified plasmid r65
  2. Fragmented it mechanically (random break points) into linear sub-fragments
  3. Used a heater treatment — crack cell walls of e.coli
  4. Used a low DNA:cell ratio
  5. Grew bacteria in a solid medium with antibiotic
  6. Clinal colony is formed
29
Q

Why must the bacteria be grown in a solid medium with an antibiotic when modifying a plasmid for particle purposes

A

By plating it on a solid medium, individual survivors are physically separated and can form a clinal colony, with all cells having the same plasmid

30
Q

Why must you use a heater treatment when modifying the plasmids for particle purposes

A

Because you need to crack open the walls on the E.Coli, so they can be introduced to the bacteria and allow an uptake of DNA from the medium

31
Q

What are the 3 steps in molecular cloning

A
  1. Modify your plasmid
  2. Physical mapping
  3. Vector usage
32
Q

Why should you use physical mapping when cloning DNA

A

To map different functions of internal structures within the plasmid to different segments.
Helps determine specific antibiotic resistance genes.

33
Q

What two main enzymes make recombining DNA possible

A

Restriction endonuclease and DNA ligase

34
Q

How did scientist manage to express a eukaryotic DNA sequence in bacteria using plasmid as a vector (using African clawed frog DNA)

A
  1. The isolated the rRNA genes from the genomic DNA
  2. They cut the genes with EcoR1 (restriction endonuclease)
  3. DNA from the frog had the same sticky ends as the plasmid
  4. Cut the plasmid with EcoR1
  5. Located fragments with the plasmid
  6. Reintroduced plasmids back into E.Coli
35
Q

What do cloning vectors allow you to do

A
  • they’re capable of replication
  • bear a selectable marker
  • incorporate a DNA fragment of interest to the experimenter
  • there’s many types based off of different replication systems
36
Q

What does restriction-ligation allow you to do

A
  • fragment DNA in predictable planes

- recombine with ligase into new molecules

37
Q

What does molecular cloning allow you to do

A
  • isolate and test molecules separately
  • introduce into bacteria a low DNA:solvent ratio
  • create surviving colonies of a single bacterium with a single DNA molecule
38
Q

When would you use a plasmid vector

A

For molecules of about 20 bases

They’re very versatile and popular but low limit in size

39
Q

When would you use a phage vector

A

For about 50 bases - bigger in size

For storing and modifying larger fragments

40
Q

When would you use a bacterial artificial chromosome

A

For big special plasmids

With special origin of replication

41
Q

How come your labelled probe binds in a stable manner and not all the probes, even if they have sequence matching

A

The probe you want to bind has been immobilised under hybridisation conditions, including a high temp and with a concentration of formamide. So it will form lots of and enough hydrogen bonds to bind in a stable manner

Genetics And Evolution (7 decks)

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