DNA

Question 1

Describe a primer

Answer 1

18 bases long Labelled 5’ and 3’ ends

Question 2

What is the new DNA profiling technique?

Answer 2

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

Question 3

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

Answer 3

• 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

Question 4

What is the use for DNA fingerprinting?

Answer 4

Immigration, paternity, murder, identification

Question 5

What is the first step in primer synthesis?

Answer 5

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

Question 6

When is a DNA profile identical?

Answer 6

If twin or clone

Question 7

What must be considered for primer design?

Answer 7

Dimerisation and self-complementing

Question 8

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

Answer 8

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

Question 9

How do you make an agarose gel?

Answer 9

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

Question 10

What primer is used for DNA fingerprinting?

Answer 10

Tag on the 5’ to prevent interference with replication

Question 11

What are the good points of primer design?

Answer 11

Solid phase

Automated

Can attach probe to either end

Question 12

What is the second step in primer synthesis?

Answer 12

Oxidation of phosphate group by I₂

Question 13

What dyes are used for gel electrophoresis?

Answer 13

Xylene cyanol ff and Bromophenol blue

Ethidium bromide

Question 14

What are the advantages of pyrosequencing?

Answer 14

Light detected by luminometer

Automated

Real time

Question 15

What bond links the base to the nucleotide?

Answer 15

A glycosidic bond

Question 16

Describe southern blotting

Answer 16

Cut locus with enzyme.

Separate on size with gel and see fluorescent for DNA profile

Question 17

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

Answer 17

One peak = XX = female

Question 18

What is DNA polymerase?

Answer 18

Used to join nucleotides to DNA,

PCR = thermo equatis

Question 19

What is the third step in primer synthesis?

Answer 19

Cl2CHCO2H removal of DMT

Question 20

Describe DNA origami

Answer 20

Parallel duplexes held in place by staple strands.

Question 21

Describe DNA walker II

Answer 21

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

Bends to adjacent hinges and ligases with ATP.

Cleavage by RE.

Question 22

What is the final step in primer synthesis?

Answer 22

Concentrated NH3 removal of the resin

Question 23

What are the fluorescent probes used in new DNA profiling?

Answer 23

3 NED = yellow

4 FAM = blue

3 JOE = green

Question 24

What is the structure of the capillary electrophoresis set up?

Answer 24

Argon laser - 500nm

Formaldehyde to break DNA into single strands

Question 25

How many rounds of PCR are performed?

Answer 25

N rounds = 2^N Usually 20-30 rounds

Question 26

What does comparing DNA fingerings allow?

Answer 26

Identify parents/murderer and shows mutations in offspring

Question 27

What is the structure of DNA?

Answer 27

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

Question 28

What are the steps of pyrosequencing?

Answer 28

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

Question 29

Describe DNA walker I

Answer 29

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.

Question 30

What are the three steps of DNA polymerase reaction?

Answer 30

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

Question 31

What is investigated in DNA fingerprinting?

Answer 31

Hypervariable regions, which differ by length in each person. `Uses fluorescent tag Microsatellite = 2-4bp Minisatellite = 10s of bps

Question 32

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

Answer 32

More abundant, showed neanderthals arent our ancestors

Question 33

What is the starting point for primer synthesis?

Answer 33

Base attached at to resin at 3' end

Question 34

What is the structure dimethoxytrityl phosphoramidite?

Answer 34

Question 35

What are the three forms of DNA?

Answer 35

A: Squat, less DNA B: Normal Z: Left handed helix

Question 36

What is the protocol for gel electrophoresis?

Answer 36

5ul mix plus sucrose and dye Charge separates on size, pulls to cathode identify by dye/UV light

Question 37

What are the enzymes used for pyrosequencing?

Answer 37

1, DNA polymerase 2. Sulfurylase 3. Luciferase and Luciferin 4. Apyrase

Question 38

Give the stages to the hamiltonian problem

Answer 38

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)

Question 39

Describe the set up of the PCR

Answer 39

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

Question 40

Which bases pair? Which are two rings?

Answer 40

GC and AT AG = purines = 2 rings

Question 41

Whats the reagent of the DNA polymerase reaction?

Answer 41

Primer, dNTP and DNA polymerase