CHAPTER 5 Flashcards
(86 cards)
1
Q
ISOLATE GENETIC MATERIAL FROM THE CELL
A
DNA and RNA extraction
2
Q
separate genetic material from unclotted substances that may interfere PCR amplification
A
DNA and RNA extraction
3
Q
DNA and nucleic acid that can be extracted
A
- Plasmid DNA
- Viral nucleic acids
- Genomic DNA from Blood and Biological fluids
- Genomic DNA from Tissue and Cells
- Genomic DNA from forensic samples
- Genomic DNA from plants and fungi
- Genomic DNA from food and feed
- Ancient DNA
4
Q
3 basic steps of DNA and RNA extraction
A
- lysis
- precipitation
- purification
5
Q
the cell and the nucleus are broken open to release the DNA inside
A
lysis
6
Q
separates DNA from cellular debris
A
precipitation
7
Q
organic detergent or enzymes used in lysis
A
- NaOH
- SDS (Sodium Dodecyl Sulfate)
8
Q
inorganic detergent or enzymes used in lysis
A
- Tris - EDTA
- EDTA
- SDS ( Sodium Dodecyl sulfate)
9
Q
reagents used in precipitation
A
- Na+ ions
- Ethanol/Isopropanol
10
Q
neutralize the negative charges on the DNA molecule which makes them more stable and less water soluble
A
Na+ ions
11
Q
added and causes DNA to precipitate out of the aqueous solution because it is not soluble in alcohol
A
Ethanol or Isopropanol
12
Q
what is used to rinse DNA to remove any remaining unwanted material and cellular debris
A
Alcohol
13
Q
designed to lyse outer cell membrane, but will not break down nuclear membrane
A
Cell lysis buffer
14
Q
examples of cell lysis buffer
A
- non-ionic detergent
- salt
- buffer
- EDTA
15
Q
remove contaminating protein
A
phenol
16
Q
remove most of the protein by digesting with proteolytic enzymes
A
proteinase k
17
Q
prevents cutting of DNA during isolation. It solubilizes lipids and a lot of protein to remove them from the DNA
A
Chloroform
18
Q
chelating of Mg2+
A
EDTA
19
Q
cofactor of DNAse
A
Mg2+
20
Q
is an anionic detergent which helps cell membrane and nuclear envelope to break open. Inhibit RNAse and DNAse (denatures protein)
A
Sodium Dodecyl Sulfate
21
Q
prevent DNA and RNA degradation
A
Tris - EDTA buffer
22
Q
Used for final resuspension and storage because it preserves any changes in pH
A
Tris-EDTA buffer
23
Q
reagent for precipitation of DNA for organic procedure
A
Ethanol
24
Q
reagent for precipitation of DNA for inorganic procedure
A
Isopropanol
25
reagent for precipitation of DNA
Ethanol and Isopropanol
26
different types of DNA extraction method
1. Chemical based DNA extraction method
2. Solid phase DNA extraction method
27
2 types of Chemical based DNA extraction method
* Organic solvent-based DNA extraction
* Inorganic solvent-based DNA extraction
28
2 types of inorganic solvent-based DNA extraction
- salting out method
- proteinase k method/enzymatic method
29
principle is based on the difference in the solubility between H2O and organic solvents
Phenol-Chloroform method
30
make use of high salt concentration to precipitate proteins (DNA will remain in the salt)
salting out method
31
advantage of salting out method
- non-toxic
- simple to perform
- inexpensive
32
advantage of phenol-chloroform method
- very high quality DNA
- effective in removing proteins and lipids
33
disadvantage of phenol-chloroform method
- toxic (special handling is required)
- time consuming
34
silica column based DNA extraction method
solid phase DNA extraction method
35
utilizes silica in the presence of salts (disrupts hydrogen bonding in H2) that will allow H2O to adhere in silica membrane
solid phase DNA extraction method
36
advantage of solid phase DNA extraction method
rapid denaturation of nucleases and stabilization of RNA
37
drawbacks of organic extraction
- laborious and manually intensive processing
- difficult method
38
what is the process fo organic DNA extraction (Phenol Chloroform Method)
1. Lyse/ Homogenize cells
2. Acidification
3. Extraction
4. Precipitation
39
what is the top layer of phenol chloroform method
aqueous solution with precipitated DNA
39
what is the basis of phenol-chloroform method
density of DNA in water
40
what is the bottom layer of the phenol chloroform method
protein contaminants and cellular debris
41
reagents used in lysis in phenol-chloroform method
- NaOH
- SDS (sodium dodecyl sulfate)
42
reagents used in acidification in phenol-chloroform method
acetic acid and salt are added to neutralize the solution
43
reagents used in extraction in phenol-chloroform method
phenol and chloroform
44
reagents used in DNA precipitation in phenol-chloroform method
ethanol
45
DNA will be pipetted for precipitation with ethanol
Supernatant
46
Phenol and chloroform will extract the proteins
Bottom layer in phenol-chloroform method
47
added to prevent RNA contamination
RNAse
48
process in inorganic DNA extraction method or salting out method
1. lysis
2. protein precipitation
3. DNA precipitation
49
reagents used in lysis in salting out method
- Tris EDTA
- EDTA
- SDS (sodium dodecyl sulfate)
50
reagent that maintains pH
Tris EDTA
51
chelates Mg2+
EDTA
52
detergent (denatures protein)
sodium dodecyl sulfate
53
reagents used in protein precipitation in salting out method
sodium acetate
54
allow proteins to clump together for easier removal
sodium acetate
55
reagents used in DNA precipitation in salting out method
isopropanol
56
what helps in the DNA precipitation for the salting out method
- sodium acetate
- sodium chloride
- potassium acetate
- ammonium acetate
57
facilitates high DNA yield but it is time consuming
proteinase K DNA extraction method
58
t/f: if not maintained well in a cold chain, the proteinase K cannot be utilized for a longer period of time
T
59
t/f: the lower stability of the enzyme in proteinase K method is another major issue in this method
T
60
process in silica column based DNA extraction method
1. lyse cellsand spin to remove large cell debris
2. apply lysate reagent to silica column (spin column) or beads
3. addition of alcohol
4. microcentrifugation or nucleic acid extractor machine
5. adsorbed DNA will be washed using buffers
6. final product elute DNA
61
principle is based on the unique chemistry of interaction between silica and DNA
Silica-based DNA extraction method
62
silica particles (column or beads) bind or adsorbed with DNA during centrifugation especially in high salt concentration
Silica- based DNA extraction method
63
cation chelating resin binds to the negatively charged phosphate of DNA and helps in the extraction fo DNA
CHELEX
64
utilizes a cation-chelating resin which binds to the negatively charged phosphate of DNA and helps in the extraction of DNA
DNA extraction using anionic resin
65
rapid and used in forensic
DNA extraction using anionic resin
66
process of DNA extraction using anionic resin
1. cell lysis
2. add chelex resin
3. boil at 100'C to break open the cell to release DNA sample
4. centrifuge
5. Transfer and retain DNA extract
67
positively charged magnetic beads attract the negatively charged DNA. The DNA is separated under the magnetic field
DNA extraction by magnetic beads
68
process of DNA extraction by magnetic beads
1. add lysis buffer
2. bind DNA and wash away cellular debris
3. elute DNA from magnetic beads
4. transfer and retain DNA extract
69
a procedure for separating particles (such as viruses or ribosomes or molecules such as DNA), in which the sample is placed on a preformed gradient such as sucrose or caesium chloride
Density gradient method
70
reagent used to separate DNA from different blood components in density gradient method
Ficoll
71
how long is extracted DNA stored for long-term storage
-20 to - 80'C for months to years
72
how long is extracted DNA stored for short-term storage
4'C
73
requires strict precautions to avoid sample degradation
RNA isolation
74
goal: prevent degradation by RNAses
RNA isolation
75
what is used to wash materials in RNA isolation
- DEPC-treated water
- NaOH
- H2O2
76
how to convert reagent to RNAse free reagents
heat treatment
77
strong denaturant of RNAses and can be used instead of high-salt buffers
Guanidine Isothiocyanate (GITC)
78
T/F: RNA is precipitated by addition of two volumes of ethanol or one volume of isopropanol
T
79
top layer of organic extraction of total RNA
aqueous solution with total RNA
80
bottom layer of organic extraction of total RNA
proteins contaminants solvent
81
process in organic extraction of total RNA
- lysis
- extraction
- RNA precipitation
82
reagent for lysis of organic extraction of total RNA
Guanidinium isothiocyanate
83
reagent for extraction of organic extraction of total RNA
phenol and chlorroform
84
reagent for RNA precipitation of organic extraction of total RNA
ethanol
85
silica columned based method for RNA process
- lysis
- RNA adsorption
- wash RNA
- elute RNA
