DNA Flashcards
Describe a primer
18 bases long Labelled 5’ and 3’ ends
What is the new DNA profiling technique?
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
What are problems with ancient DNA? How is it overcome?
- 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
What is the use for DNA fingerprinting?
Immigration, paternity, murder, identification
What is the first step in primer synthesis?
MDT phosphoramidite with base attaches to base, leaving group leaves MDT
When is a DNA profile identical?
If twin or clone
What must be considered for primer design?
Dimerisation and self-complementing
What is the structure of DNA? (Which end is which?)
5’ - ribose - nucleotide off 1’ C - phosphate - 3’
How do you make an agarose gel?
1-3% agarose plus water, microwave, add to mould with comb
What primer is used for DNA fingerprinting?
Tag on the 5’ to prevent interference with replication
What are the good points of primer design?
Solid phase
Automated
Can attach probe to either end
What is the second step in primer synthesis?
Oxidation of phosphate group by I2
What dyes are used for gel electrophoresis?
Xylene cyanol ff and Bromophenol blue
Ethidium bromide
What are the advantages of pyrosequencing?
Light detected by luminometer
Automated
Real time
What bond links the base to the nucleotide?
A glycosidic bond
Describe southern blotting
Cut locus with enzyme.
Separate on size with gel and see fluorescent for DNA profile
How can you determine if male/female with newer DNA profiling?
One peak = XX = female
What is DNA polymerase?
Used to join nucleotides to DNA,
PCR = thermo equatis
What is the third step in primer synthesis?
Cl2CHCO2H removal of DMT
Describe DNA origami
Parallel duplexes held in place by staple strands.
Describe DNA walker II
Hinges (4bp hinge) bind walker by sticky ends.
Bends to adjacent hinges and ligases with ATP.
Cleavage by RE.
What is the final step in primer synthesis?
Concentrated NH3 removal of the resin
What are the fluorescent probes used in new DNA profiling?
3 NED = yellow
4 FAM = blue
3 JOE = green
What is the structure of the capillary electrophoresis set up?
Argon laser - 500nm
Formaldehyde to break DNA into single strands
How many rounds of PCR are performed?
N rounds = 2^N
Usually 20-30 rounds
What does comparing DNA fingerings allow?
Identify parents/murderer and shows mutations in offspring
What is the structure of DNA?
Antiparallel right-handed helix. 10bp per turn and 2nm width.
What are the steps of pyrosequencing?
- Primer + dNTP -> Primer + PPi (if complementary)
- PPi + AMP -> ATP
- ATP -> Light
- dNTP -> dNMP + PPi
Describe DNA walker I
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.
What are the three steps of DNA polymerase reaction?
- Melt (94C) - break strands
- Anneal (55C) - join primer to strands
- Extension (72C) - 3’ OH attack phosphate, release PP
What is investigated in DNA fingerprinting?
Hypervariable regions, which differ by length in each person.
`Uses fluorescent tag
Microsatellite = 2-4bp Minisatellite = 10s of bps
Why is mitochondria DNA sometimes used? What did it show?
More abundant, showed neanderthals arent our ancestors
What is the starting point for primer synthesis?
Base attached at to resin at 3’ end
What is the structure dimethoxytrityl phosphoramidite?
What are the three forms of DNA?
A: Squat, less
DNA B: Normal
Z: Left handed helix
What is the protocol for gel electrophoresis?
5ul mix plus sucrose and dye
Charge separates on size, pulls to cathode identify by dye/UV light
What are the enzymes used for pyrosequencing?
1, DNA polymerase
- Sulfurylase
- Luciferase and Luciferin
- Apyrase
Give the stages to the hamiltonian problem
- mix cities and flights in solution
- Ligase = mass parallel processing
- PCR select ones with start and finish cities
- isolate by length, select correct
- use magnet probe of city, remove all which don’t magnetise
- Sequence final strand(s)
Describe the set up of the PCR
Thermocycler well, in tube with mix and mineral oil layer on top
Which bases pair? Which are two rings?
GC and AT
AG = purines = 2 rings
Whats the reagent of the DNA polymerase reaction?
Primer, dNTP and DNA polymerase
