Revision Oral

Question 1

Steps involved in DNA extraction

Answer 1

1 - Tissue disruption
2 - Cell disruption
3 - Removal of Cellular components
4 - Seperation of DNA from lysate
5 - Isolation of pure DNA

Question 2

1. Tissue disruption

Answer 2

Grinding for bone
Pulverisation of muscle

Question 3

2. Cell disruption

Answer 3

An SDS detergent is used to break the cell membrane by disrupting the lipid bilayer.
Guanidinium is used to break cell membranes by destabilising hydrophobic interactions.

Question 4

3. Removal of cellular components

Answer 4

SDS denatures proteins and lipids.
Proteinase-K digestion then occurs followed by protein precipitation to remove proteins.
Heat is used to denature proteins.
Phenol extraction denatures proteins.

Question 5

4. Separation from lysate (organic extraction, salting out and binding to column)

Answer 5

For organic extraction, DNA is within aqueous phase and can be precipitated.
For salting out, proteins precipitate at high salt concentrations and can be removed, leaving DNA.
For binding to column, DNA is bound to a solid support while lysate is removed.

Question 6

5 - concentration of pure DNA

Answer 6

Ethanol or isopropanol and salt causes precipitation of DNA.
DNA is bound while lysate is washed away. Pure DNA is then eluted into a buffer.

Question 7

EDTA

Answer 7

EDTA is present in most buffers for DNA extraction and acts as a chelating agent for magnesium ions.
It helps to inactivate the nuclease during the DNA extraction process.

Question 8

Methods of DNA purification

Answer 8

1 - organic extraction
2 - Chelex resin
3 - FTA paper
4 - Qiagen kits
5 - puregene

Question 9

Organic extraction

Answer 9

Liquid is separated into 3 layers. Bottom one is the organic layer & contains phenol that’s toxic. Middle one contains denatured proteins and other cell components. Top layer contains DNA.
+large quantity of pure DNA obtained
-time consuming, hazardous

Question 10

Chelex resin

Answer 10

Cell suspension is boiled in a buffer with 5% chelex. It’s centrifuged to pellet resin. DNA is ready in supernatant.
+ quick, cheap, few steps
- crude, can result in inhibition of PCR

Question 11

FTA paper

Answer 11

An absorbent paper that contains chemicals that lyse cells, denature proteins and protect nucleic acids from oxidation and UV damage.

Question 12

FTA paper 2

Answer 12

Spot cells onto paper and wash in solvent to remove haem and other PCR inhibitors. Rinse in a buffer and use card for PCR.
+DNA in card is stable for years, quick, simple
-Dry paper pieces jump out of PCR tubes due to static electricity

Question 13

Qiagen kits

Answer 13

1. Cell lysis using quanidinium and proteinase K digestion.
   DNA then binds to silica membrane in column.
   Washing out the membrane to remove proteins and other contaminants. Pure DNA is eluted from the membrane.
   +high quality and yields of DNA
   -expensive

Question 14

Puregene

Answer 14

Cell lysis in buffer containing Tris, EDTA and SDS. Proteins then precipitated out using ammonium acetate. DNA precipitated out using isopropanol at -20degrees. DNA washed with 70% ethanol and dissolved in buffer.
+rapid, consistent

Question 15

DNA quantification methods

Answer 15

1 - Agarose gel electrophoresis
2 - Spectrophotometry
3 - Slot blotting
4 - Fluorescent methods eg.Picogreen assay
5 - Real time PCR

Question 16

Agarose gel electrophoresis

Answer 16

DNA migrates to positive pole in an electric field.
Large fragments move slower than small ones.
Visualised by staining with fluorescent dyes (gel red).
Loading dyes are used.
Leaders are used for sizing and quantification.

Question 17

Why are loading dyes used?

Answer 17

Colour the DNA solution, making gel easier to load.
Indicate distance travelled by DNA.
Help DNA solution sink into well

Question 18

Spectrophotometry

Answer 18

Measures absorbance of UV light (260nm) by DNA.
A260 of 1 = 50µg/ml

Question 19

Slot blotting

Answer 19

Genomic DNA is immobilised on a nylon membrane and hybridised to a probe. Measurement is made relative to a set of standards.
Can detect down to 150pg of DNA.

Question 20

Fluorescence based methods

Answer 20

Eg. Picogreen is a sensitive fluorescent dye which intercalates into dsDNA.
96 well format.
Unknown samples are compared to known standards.

Question 21

Real time PCR

Answer 21

Uses fluorescently labelled probes or dyes.
Fluorescence is measured every cycle and is proportional to the amount of PCR product amplified.
Unknown samples compared to set of known standards.
Most accurate method of DNA quantification.

Question 22

Amounts of DNA in cell, blood and hair.

Answer 22

Human cell - 6.6pg
Blood stain - up to 500 ng/cm2
Hair root - up to 750ng

Question 23

Types of genetic/ DNA markers

Answer 23

RFLP
VNTR
STR
SNP
Mitochondrial DNA
Y chromosome

Question 24

RFLP analysis

Answer 24

1 - gel electrophoresis and southern blotting of genomic DNA
2 - PCR (denaturation, annealing, extension)
3- Agarose gel electrophoresis

Question 25

VNTR analysis / DNA fingerprinting

Answer 25

1- fragment produced using restriction enzymes
2 - Run on agarose gel (smear produced)
3 - southern blotting
4 - Probe membrane with multi or single locus probe
5 - Detect probe using autoradiography

Question 26

Single and multi locus probes

Answer 26

Single locus - detects 1 locus, will only have 2 bands.
Multi locus - detects multiple sites. Lots of bands.

Question 27

Southern blotting

Answer 27

1 - DNA in gel is denatured using alkaline solution
2 - ssDNA in gel is blotted onto nylon membrane
3- DNA permanently fixed onto membrane by baking at 80 degrees or UV radiation

Question 28

STR analysis

Answer 28

• PCR to amplify
• PAGE (polyacrylamide gel electrophoresis) as it has smaller pore size
• 2 strands stay separate

Question 29

PCR products

Answer 29

Fluorescence based detection used - higher the peak = more DNA
One 5’ end labelled PCR primer is used so only one strand fluoresces

Question 30

Advantages of capillary electrophoresis

Answer 30

Minute amounts of sample required
Short separation times
Injection, separation and detection can be automated

Question 31

Difference between dNTP and ddNTP

Answer 31

Oxygen has been removed twice from ddNTP.
Absence of 3’OH prevents formation of a phosphodiester bond

Question 32

SNPs

Answer 32

Detected by RFLP, DNA microarrays or single base extension.

Question 33

DNA microarray

Answer 33

An ordered grid of oligonucleotides at fixed positions on silicon.
Allows simultaneous typing of thousands of SNPs

Question 34

Single base extension (mini sequencing)

Answer 34

PCR carried out using primers that anneal adjacent to the SNP. A fluorescent ddNTP is used.

Question 35

Y chromosome

Answer 35

Contains STRs and SNPs
Analysed using standard methods for snp/str

Question 36

Sex Identification

Answer 36

Uses the amelogenin gene present on sex chromosomes. The length is used to distinguish sex. 106bp on X chromosome, 112bp on Y chromosome.

Question 37

PCR process

Answer 37

Denaturation - heated to separate strands
Annealing - cooled so primers can form hydrogen bonds with target sequence
Extension - DNA polymerase binds to sequence and adds nucleotides to the 3’ end

Question 38

Gel electrophoresis basics

Answer 38

Gel creates a mesh that DNA fragments travel through
Pore size increases as gel size decreases