MIDTERM: DNA ISOLATION

Question 1

• Key to the molecular biology
• Removal of nucleic acids (DNA and/or RNA) from the cells in which they normally reside.

Answer 1

Nucleic Acid Extraction

Question 2

APPLICATIONS OF NUCLEIC ACID EXTRACTION

Answer 2

DIAGNOSING DISEASE AND GENETIC DISORDERS
- FORENSICS
- PATERNITY TESTS
- ANCESTRY TRACKING
- GENETIC ENGINEERING

Question 3

Target Nucleic Acids: free from contamination with macromolecules such as:

Answer 3

PROTEINS, CARBOHYDRATES, LIPIDS OR OTHER NUCLEIC ACIDS

Question 4

isolated the nuclein (DNA) from the WBCs he obtained from the pus on collected surgical bandages in a nearby hospital.

Answer 4

Friedrich Miescher (1869)

Question 5

process where the substance settle at the bottom of tube (the DNA)

Answer 5

PRECIPITATION

Question 5

Developed from density-gradient centrifugation strategies

Answer 5

Early Routine Laboratory Procedures of DNA Isolation

Question 6

demonstrate the semi-conservative replication of DNA

Answer 6

 Messelson & Stahl (1958)

Question 7

daughter DNA consists is consist of old and new strand, preserve the integrity of the parent strand

Answer 7

Semiconservative

Question 8

• Can be lysed by high pH (alkaline) and detergents
• Break/lyse the cell wall by adding detergent or high pH

Answer 8

A. Bacteria and Fungi

Question 9

thin cell layer

Answer 9

Gram +

Question 10

thick cell layer of peptidoglycan (

Answer 10

Gram -

Question 11

Some bacteria with tough cell walls:
- uses the enzyme “lysozyme”

Answer 11

ENZYMATIC DIGESTION

Question 12

grinding/vigorously mixing with glass beads
- Grind the thick cell wall, physically break open the bacteria cell)

Answer 12

MECHANICAL METHOD

Question 13

detergent (1% sodium dodecyl sulfate/SDS) and a strong base (0.2M NaOH) in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA; anticoagulant) and glucose.

Answer 13

CHEMICAL LYSIS

Question 14

commercial reagents designed for isolation of DNA in amplification procedures (PCR)

Answer 14

YEAST FILAMENTOUS FUNGI & GRAM + BACTERIA

Question 15

o Held within free viruses or integrated into the host genome along with host DNA.
o Cell-free specimens (plasma) will be used for viral detection
o May require concentration of viroids by centrifugation or other methods

Answer 15

Viral DNA

Question 16

naked virus, no protein capsule basic structure of virus

Answer 16

VIROIDS

Question 17

o Obtained from the blood plasma
o Purified of red blood cells (RBCs) & other components by either differential density-gradient centrifugation or differential lysis

Answer 17

Nucleated Cells in Suspension (Blood & Bone Marrow Aspirates)

Question 18

 Exosomes (release by cancer cells)
o Small vesicles (30 – 100 nm in diameter), which form by imagination & budding from the inside of cellular endosome vesicles and are secreted by living cells
o Contain nucleic acid and can be collected through centrifugation
o Diagnostic & prognostic analyses purposes (liquid biopsy)
o Solid-phase collection of nucleic acid

Answer 18

Plasma

Question 19

Dissolved by mincing the tissue

Answer 19

FRESH TISSUE

Question 20

Dissociated by grinding and homogenizing the tissue

Answer 20

FROZEN TISSUE

Question 21

• preserve tissue morphology
• May be deparaffinized by soaking in XYLENE and tissue is rehydrated by soaking it in decreasing concentrations of ethanol
• May be used directly without dewaxing (high contamination if not dewaxed)
• tissue fixatives affect the quality of DNA

Answer 21

FIXED EMBEDDED TISSUE

Question 22

mercury based fixatives that can damage DNA

Answer 22

B-5 and BOUIN’S

Question 23

DNA ISOLATION CHEMISTRIES
* uses a combination of HIGH SALT, and an ORGANIC MIXTURE of PHENOL & CHLOROFORM
* Readily dissolves hydrophobic contaminants (lipids & lipoproteins)
* collect cell debris & strips away most DNA – associated proteins

Answer 23

ORGANIC ISOLATION METHODS

Question 24

* Does not include the organic extraction step * Sometimes called “SALTING OUT”, makes use of LOW pH and high salt conditions to selectively precipitate proteins, leaving the DNA in the solution * Excess salt is removed by rinsing the pelted nucleic acid in 70% ethanol, centrifuging & discarding the supernatant, then dissolving the DNA pellet in rehydration buffer (10 mM, Tris, ImM EDTA, or water)

Answer 24

INORGANIC ISOLATION METHOD

Question 25

* Used SOLID MATRICES (silica-based products) in the form of columns or beads to bind & hold the DNA for washing

Answer 25

C. SOLID PHASE ISOLATION

Question 26

NON – INVASIVE HUMAN DNA ISOLATION

Answer 26

1. GENOMIC DNA PURIFICATION FROM HAIR 2. GENOMIC DNA PURIFICATION FROM SALIVA 3. GENOMIC DNA EXTRACTION FROM URINE SAMPLE

Question 27

→ Hair sample is incubated (95°C) for 10 mins in NaOH (strong base/alkali) buffer & the supernatant is subjected to DNA purification after centrifugation

Answer 27

Simple Alkaline Lysis Methods (root of hair)

Question 28

→ Hair sample is incubated (56°C) for 2 hours with buffer containing Tris= HCL, EDTA, NaCl, SDS, DTT and proteinase K, followed by gentle mixing and incubation (60 °C) for 2 hours or until the hair dissolved completely and DNA can be extracted from the solution

Answer 28

Smooth Chemical Digestion Method using Dithioreitol (DTT)

Question 29

cells found in the saliva, exfoliated buccal epithelial cells and other cells

Answer 29

GENOMIC DNA PURIFICATION FROM SALIVA

Question 30

→ Suspended in Lysis buffer which include Tris, EDTA, SDS & proteinase K → Incubated (56°C) for 1-3 hours until the tissue is totally dissolved and DNA

Answer 30

Buccal Swabs

Question 31

→ Samples from saline rinse need to be processed/frozen immediately after collection → To inhibit growth of bacteria → Alcohol containing mouthwash must be used (kills microorganism and reduce bacterial growth)

Answer 31

Mouthwash Method

Question 32

- Inverted and swirled in a specimen cup to create a homogenous suspension of cells followed by the centrifugation - Supernatant is removed and a dry pellet containing cells is chilled (20°C) for 15 mins. Followed by the addition of Lysis buffer (Tris, EDTA, SDS, Proteinase K) - Sample is incubated (56°C) for 2 hours and then DNA is extracted from solution

Answer 32

GENOMIC DNA EXTRACTION FROM URINE SAMPLE

Question 33

PROTEOLYTIC LYSIS OF FIXED MATERIAL

Answer 33

* PARAFFIN – EMBEDDED SPECIMENS * BEFORE LYSIS * REAGENTS FOR CELL LYSIS

Question 34

o Dewaxed w/ XYLENE specimens o Rehydrated before nucleic acid isolation

Answer 34

PARAFFIN – EMBEDDED SPECIMENS

Question 35

o Cells may be washed by suspension and centrifugation in saline/other isotonic buffers

Answer 35

BEFORE LYSIS

Question 36

o Simple screens: detergents (SS or triton) o PCR amplification: mixture of this buffer and proteinase K (capable of breaking down proteins)

Answer 36

REAGENTS FOR CELL LYSIS