DNA Methods

Question 1

DNA Extraction + Purification Method

Answer 1

Cells lysed using Tris-Cl and EDTA. Proteases degrade cellular proteins. DNA binds membrane stronger the centrifugal force, other substances settle to bottom through membrane. Wash container to remove contaminants. Elution solution is added to tube DNA unbinds from membrane and settles into tube

Question 2

Nanodrop Spectrophotometer Outline

Answer 2

Used to calculate nucleic concentration of an extracted sample. Passes wavelength of light through tube and measures absorbance. Beer-Lambert equation. Higher absorbance = higher conc. Can evaluate purity

Question 3

Qubit Fluorometer Outline

Answer 3

Fluorescent molecule binds ds DNA + reaches excited state. higher fluroescent conc = higher DNA conc (known conc used to calibrate). More accurate quantification then Nanodrop but can’t evaluate purity

Question 4

Gel Electrophoresis Outline

Answer 4

Separation and analysis of macromolecules based on charge and size (DNA = negatively charged (phosphate backbone)). Differentiates nucleic acids. Size analysed based on column of fragments of known size. DNA gels: polyacrylamide and agarose

Question 5

Ethidium Bromide (EtBr) Outline

Answer 5

Chemical dye that binds with ds DNA/RNA by filling gaps in nucleic acids. Fluoresces red/brown when UV light shines on it. Warning; mutagen

Question 6

Restriction Enzymes Outline

Answer 6

Molecular scissors cut DNA at selective regions (specific target site). Isolated from bacteria. Used in molecular cloning (in vectors). 2 types; blunt ends (straight vertical cut) and sticky ends (diagonal)

Question 7

Which Restriction Enzyme Cut is Most Beneficial

Answer 7

Sticky ending. DNA bases are exposed, easier editing

Question 8

Molecular Cloning Outline

Answer 8

Cell lysed and Human gene isolated. Bacteria plasmid extracted from bacterial cell. Restriction enzymes cut gene and plasmid with complementary shapes. DNA ligase combines plasmid + human gene to form recombinant plasmid

Question 9

Hybridization Outline

Answer 9

2 complementary single strand RNA +/ DNA molecules bind together to form a double stranded molecule (complementary base pairing)

Question 10

Oligonucleotide Probes Outline

Answer 10

Binds to complementary nucleotide sequence (A, T/U, G & C). Behave like natural nucleotide fluoresce. Useful for qPCR and Southern Blotting

Question 11

Southern Blotting Outline

Answer 11

Shows fragments of expected size, can’t verify sequence type (can’t identify SNPs). Membrane made of nitrocellulose (positive). Single stranded probes hybridize to sequence of interest. dsDNA denatured using alkaline solution (not heat)

Question 12

Restriction Fragment Length Polymorphism (RFLP) Analysis Method

Answer 12

Extract DNA fragment from sample. Digest DNA restriction enzymes. Segments separated by gel electrophoresis. Transfer fragments to Southern blot. Hybridized DNA sequence with fluroescence. Band patterns indicate polymorphisms

Question 13

Theory RFLP is Based on

Answer 13

Mutations in DNA at recognition site means restriction enzymes can’t cut fragments in specific places. If fragment is cut correctly it won’t be the same size as non-muatated (non-diseased fragment) and won’t align with pattern. aditionally specific fluroescent probe won’t bind so specififc wavelength won’t be picked up

Question 14

Fluorescence In Situ Hybridization (FISH) Outline

Answer 14

Fluorescently labelled single stranded probes. Used to detect large chromosomal deletions, repeats and translocations. Used on uncultured cells, high accuracy, rapid results (1-2 days)

Question 15

Array Comparative Genomic Hybridization (ACGH) Outline

Answer 15

Microarray to evaluate number of copy variants. Measures amount of DNA in a sample vs the control both stained with fluroescent dyes of different colours. Deficiency: well the colour of control, Excess: well the colour of sample. Can evaluate SNPs, gene expression (in developmental diseases eg Downe’s Syndrome) and CNVs