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Natural Sciences Bath Final Year > DNA > Flashcards

DNA Flashcards

1
Q

Describe a primer

A

18 bases long Labelled 5’ and 3’ ends

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2
Q

What is the new DNA profiling technique?

A

PCR and pairs of primers for 10 loci on different chromosomes.

Plus 11th amelogenin gene (smaller in X)

Primer is 5’ labelled.

Loci are amplified with PCR Capillary electrophoresis to sort

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3
Q

What are problems with ancient DNA? How is it overcome?

A
  • DNA decays overtime (hydrolytic, oxidative, and methylated)
  • Low amount - use lots of tissue
  • Contamination - use RE to cleave bacteria CG rich segments. C is protected in hominids by methylation.
  • Low yield - change purification steps
  • Sequencing is slow - use pyrosequencing
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4
Q

What is the use for DNA fingerprinting?

A

Immigration, paternity, murder, identification

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5
Q

What is the first step in primer synthesis?

A

MDT phosphoramidite with base attaches to base, leaving group leaves MDT

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6
Q

When is a DNA profile identical?

A

If twin or clone

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7
Q

What must be considered for primer design?

A

Dimerisation and self-complementing

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8
Q

What is the structure of DNA? (Which end is which?)

A

5’ - ribose - nucleotide off 1’ C - phosphate - 3’

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9
Q

How do you make an agarose gel?

A

1-3% agarose plus water, microwave, add to mould with comb

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10
Q

What primer is used for DNA fingerprinting?

A

Tag on the 5’ to prevent interference with replication

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11
Q

What are the good points of primer design?

A

Solid phase

Automated

Can attach probe to either end

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12
Q

What is the second step in primer synthesis?

A

Oxidation of phosphate group by I2

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13
Q

What dyes are used for gel electrophoresis?

A

Xylene cyanol ff and Bromophenol blue

Ethidium bromide

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14
Q

What are the advantages of pyrosequencing?

A

Light detected by luminometer

Automated

Real time

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15
Q

What bond links the base to the nucleotide?

A

A glycosidic bond

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16
Q

Describe southern blotting

A

Cut locus with enzyme.

Separate on size with gel and see fluorescent for DNA profile

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17
Q

How can you determine if male/female with newer DNA profiling?

A

One peak = XX = female

18
Q

What is DNA polymerase?

A

Used to join nucleotides to DNA,

PCR = thermo equatis

19
Q

What is the third step in primer synthesis?

A

Cl2CHCO2H removal of DMT

20
Q

Describe DNA origami

A

Parallel duplexes held in place by staple strands.

21
Q

Describe DNA walker II

A

Hinges (4bp hinge) bind walker by sticky ends.

Bends to adjacent hinges and ligases with ATP.

Cleavage by RE.

22
Q

What is the final step in primer synthesis?

A

Concentrated NH3 removal of the resin

23
Q

What are the fluorescent probes used in new DNA profiling?

A

3 NED = yellow

4 FAM = blue

3 JOE = green

24
Q

What is the structure of the capillary electrophoresis set up?

A

Argon laser - 500nm

Formaldehyde to break DNA into single strands

25
Q

How many rounds of PCR are performed?

A

N rounds = 2^N

Usually 20-30 rounds

26
Q

What does comparing DNA fingerings allow?

A

Identify parents/murderer and shows mutations in offspring

27
Q

What is the structure of DNA?

A

Antiparallel right-handed helix. 10bp per turn and 2nm width.

28
Q

What are the steps of pyrosequencing?

A
  1. Primer + dNTP -> Primer + PPi (if complementary)
  2. PPi + AMP -> ATP
  3. ATP -> Light
  4. dNTP -> dNMP + PPi
29
Q

Describe DNA walker I

A

Walks on SS DNA Toes and heel compete for space, if heel is loose foot may peel.

Exposed strands pulled by fuel strand, which breaks it next and is peeled by second fuel strand.

Foot goes down in same, or forward position.

30
Q

What are the three steps of DNA polymerase reaction?

A
  1. Melt (94C) - break strands
  2. Anneal (55C) - join primer to strands
  3. Extension (72C) - 3’ OH attack phosphate, release PP
31
Q

What is investigated in DNA fingerprinting?

A

Hypervariable regions, which differ by length in each person.

`Uses fluorescent tag

Microsatellite = 2-4bp Minisatellite = 10s of bps

32
Q

Why is mitochondria DNA sometimes used? What did it show?

A

More abundant, showed neanderthals arent our ancestors

33
Q

What is the starting point for primer synthesis?

A

Base attached at to resin at 3’ end

34
Q

What is the structure dimethoxytrityl phosphoramidite?

A
35
Q

What are the three forms of DNA?

A

A: Squat, less

DNA B: Normal

Z: Left handed helix

36
Q

What is the protocol for gel electrophoresis?

A

5ul mix plus sucrose and dye

Charge separates on size, pulls to cathode identify by dye/UV light

37
Q

What are the enzymes used for pyrosequencing?

A

1, DNA polymerase

  1. Sulfurylase
  2. Luciferase and Luciferin
  3. Apyrase
38
Q

Give the stages to the hamiltonian problem

A
  1. mix cities and flights in solution
  2. Ligase = mass parallel processing
  3. PCR select ones with start and finish cities
  4. isolate by length, select correct
  5. use magnet probe of city, remove all which don’t magnetise
  6. Sequence final strand(s)
39
Q

Describe the set up of the PCR

A

Thermocycler well, in tube with mix and mineral oil layer on top

40
Q

Which bases pair? Which are two rings?

A

GC and AT

AG = purines = 2 rings

41
Q

Whats the reagent of the DNA polymerase reaction?

A

Primer, dNTP and DNA polymerase

Natural Sciences Bath Final Year (5 decks)

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