LAB - Nucleic Acid Extraction

Question 1

basic steps of DNA extraction/purification

Answer 1

1. cel disruption/lysis
2. purification of the nucleic acids from cellular material
3. concentration of DNA

Question 2

these disrupt non-covalent bonds in the proteins, denaturing them, and causing molecules to lose their native shape

Answer 2

detergents

Question 3

chaotropic agent

Answer 3

molecule in water solution that can disrupt the hydrogen bonding between water molecules

disrupt the intramolecular bonds that fold proteins together = denaturant; effective at disrupting RNAse

• guanidine, chloride, phenol, ethanol

Question 4

T or F. enzymatic digestion is a harsh lysis method

Answer 4

F! gentle

Question 5

proteinase K

Answer 5

• enzymatic digestion; most common
• serine protease
• cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic AAs

Question 6

lysozymes

Answer 6

• enzyme digestion; lyses gram +/- bacteria
• hydrolyzes peptidoglycan in bacterial cell wall
  >B-1,4 linkages between NAM and NAG

Question 7

Zymolase

Answer 7

enzyme digestion for yeast and fungal cell walls

Question 8

Lysostaphin

Answer 8

lyse cell walls of Staphylococci

Question 9

Lysostaphin

Answer 9

lyse cell walls of Staphylococci

Question 10

Mutanolysin

Answer 10

lyse cell walls of Listeria and other gram + bacteria

Question 11

RNAse A

Answer 11

• endonuclease
• cleaves phosphodiester bonds between 5’ ribose of a nucleotide and the phosphate group attached to the 3’ ribose of an adjacent pyrimidine nucleotide (cytosine and uracil)
• added at start or end after extraction

Question 12

DNAse

Answer 12

• endonuclease
• cleaves DNA by preferentially acting on phosphodiester bonds or adjacent to pyrimidines to produce polynucleotides with terminal 5’-phosphates

Question 13

nucleic acids can be precipitated out of solution by: (2)

Answer 13

• ethanol = routine; DNA more soluble in ethanol than isopropanol
• isopropanol = preferred for large vol extractions, less volatile than ethanol, pellets don’t stick

Question 14

carrier molecules sor co-precipitants

Answer 14

• helps with precipitating really small nucleic acid molecules
• inert substances; using alcohol and salts
• can also enhance visualization of NA pellet after precipitation
• tRNA, glycogen, linear polyacrylamide, homopolymers of nucleotide poly dA

Question 15

isolation of mitochondrial DNA

Answer 15

• enrichment = Mt DNa co-isolated with genomic DNA and is then preferentially amplified for enrichment
  > efficient
  > may introduce artifacts
• differential centrifugation
  > CsCl gradient and bis-benzamide fluorescent DNA dye
  > time consuming

Question 16

these must be lysed to extract DNA

Answer 16

cell wall and cellular membranes

Question 17

Most commercial kits use a combination of ___, ___, and ___ ___ to lyse cells

Answer 17

• enzyme
• detergents
• chaotropic agents

Question 18

nucleic acid solutions can be concentrated by “__ ___”

Answer 18

salting out; in the presence of alcohols

Question 19

mRNA can be isolated by _____ chromatography

Answer 19

affinity

> polyA tail of eukaryotic messenger RNA binds to polyT or polyU chain

Question 20

T or F. EDTA whole blood contains red and white blood cells, only the white bloods cells contain genomic DNA

Answer 20

T

Question 21

Qiagen protease is a broad-specificity ______ protease with high activity, cleaving preferentially at ____ and _____ residues.The protease digest proteins and enzymes in the sample.
Buffer AL contains a SDS. SDS is a detergent and chaotropic agent it solubilized….

Answer 21

serine
neutral and acidic
lipids and denatures and solubilizes proteins

Question 22

Buffer AL also contains ______ ______, another chaotropic salt which denatures proteins. This is also required for nucleic acid to adsorb to the silica membrane in the spin column.

Answer 22

guanidine hydrochloride

Question 23

Buffers AW1 and AW2 contain ________.These wash buffers remove ….

Answer 23

ethanol

weakly bound reagents and cellular components from the membrane

Question 24

The DNA can be eluted off the membrane using a … (Tris Cl EDTA pH 8) or molecular grade water.Tris Cl EDTA is better for long term storage as it prevents __ ________ to the DNA and the EDTA binds divalent cations which are required for _____ __________.

Answer 24

low salt buffer

pH damage

DNase activity