MOL BIO LEC DNA EXTRACTION

Miescher

1869

Who first isolated DNA from human cells in ______ by precipitation?

precipitation

Miescher first isolated DNA from human cells in 1869 by?

Density-gradient centrifugation

DNA isolation based from what type of centrifugation strategies?

Meselson and Stahl

Who demonstrated semi-

conservative replication?

Solubility differences

DNA Isolation later procedures?

Sample processing

Nucleic acid Extraction

Nucleic acid Amplification

Nucleic acid Detection

What is the basic workflow in Molecular laboratory?

high pH and detergents

Cell walls of bacteria and fungi are not thick and can be lysed by?

Gentler enzymatic methods

_______________ are less likely to damage chromosomal DNA

Larger chromosomal targets

Gentler enzymatic methods targets?

high pH 0.2M NaOH

Tris Base

EDTA

Glucose

For lysis of Bacteria and fungi cell wall what reagents is used?

1% sodium dodecyl sulfate

For lysis of Bacteria and fungi cell wall what detergent is used?

Dilute sucrose

Triton X-100

detergent

Tris buffer

EDTA

Bacteria and cell wall boil in ______ after lysozyme treatment to releases the DNA?

Single stranded

DNA extraction by boiling or alkaline

procedures becomes?

restriction enzyme analysis

Boiling of cell wall of bacteria and fungi are NOT used in?

Yeasts

Filamentous fungi

Gram + bacteria

Bacteria and Fungi

Commercial reagents (PCR)

Speed and simplicity

Advantage of Commercial reagents in Bacteria and Fungi?

Cell-free specimens (plasma)

Viral DNA is held with in________ if free virus?

Nucleated cells in suspension

Viral DNA is held with in________ if integrated in host genome

plasma

In Nucleated cells

_______ is preferred over serum?

WBCs

is where most nucleic acids come from?

Differential-gradient centrifugation

Differential lysis

WBCs are purified by?

Ficoll

In Differential-gradient centrifugation

is a highly branched sucrose polymer that does not penetrate biological membranes

Blood sample

Ficoll

In Differential-gradient centrifugation

Before centrifugation what is the order of sample in the tube? (Top to bottom)

Plasma

PMBCs

Ficoll

Granulocytes

Eythrocytes

In Differential-gradient centrifugation

After centrifugation what is the order of sample in the tube? (Top to bottom)

mononuclear cells

saline

In Differential-gradient centrifugation

Layer of __________ are extracted →

Washed by rounds of resuspension and

centrifugation with _______ → Extraction

Differential lysis

Takes advantage of the differential osmotic fragility

of RBCs and WBCs

Hypotonic buffer or water

In Differential lysis,

_________will result to the lysis of

RBCs before the WBCs

centrifugation

In Differential lysis,

WBCs are pelleted by ___________

Cells

Exosomes (Proteins, Lipid, Nucleic acids)

Nucleic acids

What are the sources of nucleic acids in plasma?

Centrifugation

Nucleic acid are collected via?

Plasma

What sample is for Diagnostics and prognosis via amplification

procedures?

Plasma

It requires procedures to concentrate the target

nucleic acid before isolation?

Solid-phase collection on beads or columns

Circulating tumor cells can be collected on

special columns

Concentrating the target nucleic acid in plasma specimen using?

100 bp or less

Tissue samples (fixed tissu)?

proteinase K

Tissue samples

Extended digestion in ________may yield longer fragments

Grind

Homogenize

Mince

Frozen tissue specimens need to?

Xylene

Fixed, embedded tissue: Deparaffinize using?

Decreasing concentration of alcohol

Fixed, embedded tissue: Rehydration using?

Buffered formalin VS Bouin's and B-5

Tissue sample

Fixatives may damage DNA to varying extent

• The type of Nucleic acid

• The source of Nucleic acid

• The type of downstream application

• The sample batch size and processing time

Four Major Factor to consider when

selecting extraction methods?

Genomic DNA

Plasmid DNA

Mitochondrial DNA

Different type of RNA

What are the types of Nucleic acid?

Commercial kit

High throughput format

Automation

What are The sample batch size and

processing time?

1. Cell lysis

2. Protein removal

3. Precipitation

4. Purification

What are the major processes of Nucleic acid extraction?

Liquid phase extraction

What kind of extraction?

Removal of cellular components

Solid phase extraction

What kind of extraction?

Removal of cellular components

Removal of contaminants and nucleic acid recovery

Alcohol precipitation

What kind of precipitation?

Removal of contaminants and nucleic acid recovery

Cell lysis

Is the process of

disintegrating the cellular

structure of the cells to

release nucleic acid into

solution?

Chemical method

Cell lysis method

Utilizes detergents and chaotropic agents to disrupt cellular membrane

and denature proteins

detergents

chaotropic agents

Chemical method utilizes ____________to disrupt cellular membrane

and denature proteins

Enzymatic method

Cell lysis method?

Uses enzymes such as proteinase K or lysozyme that disrupt cells by the

digestion of cellular structure and proteins

proteinase K or lysozyme

In Enzymatic method, uses enzymes such as _____________that disrupt cells by the

digestion of cellular structure and proteins

Physical method

Cell lysis method

Includes freeze-thaw cycles, mechanical lysis, liquid homogenization,

sonication, or high pressure to lyse cells that are hard to disrupt

Protein removal

Separation of Nucleic Acid from other cellular components?

LP

Liquid phase or Solid phase extraction method?

Organic extraction

LP

Liquid phase or Solid phase extraction method?

Inorganic extraction

SP

Liquid phase or Solid phase extraction method?

Column based extraction

SP

Liquid phase or Solid phase extraction method?

Magnetic-bead based extraction

Organic extraction

Nucleic acid extraction

combination of high salt, low pH, and an organic mixture of phenol

and chloroform

high salt

low pH

organic mixture (phenol and chloroform)

Organic extraction used combination?

Organic extraction

Nucleic acid extraction

dissolves hydrophobic contaminants.

Cetyltrimethylammonium bromide

In Nucleic acid extraction

Fungi need pretreatment with?

7-8

DNA extraction is performed at pH?

Aqueous phase

In DNA extraction, DNA remains in?

4-6

RNA extraction is performed at pH?

DNA

RNA

RNA extraction

_____ migrates to the lower organic phase or the interphase leaving only ______ in the upper aqueous phase

detergent or phenol

high salt (0.2 to 0.5 M NaCl) or RNase

inhibitors

RNA extraction

RNA isolation is performed in ________in the presence of ____________?

Guanidine isothiocyanate (GITC)

RNA extraction

is a strong denaturant of

RNases and can be used instead of high-salt buffers.

2-mercaptoethanol

Strong reducing agents such as _________may

also be added during step of RNA extraction

Ethanol

Ethanol or Propanol

Type

Denatured

Isopropanol

Ethanol or Propanol

Type

Undenatured/Pure

Ethanol

Ethanol or Propanol

Precipitation at room temperature - NO

Isopropanol

Ethanol or Propanol

Precipitation at room temperature - YES

Ethanol

Ethanol or Propanol

Amount needed to precipitate - MORE

Isopropanol

Ethanol or Propanol

Amount needed to precipitate - LESS

Ethanol

Isopropanol

Ethanol or Propanol

Viscocity at freezer temperature- VISCOUS

Inorganic extraction

sometimes called "salting out"?

Inorganic extraction

uses high concentrations of salt such as ammonium acetate, potassium acetate

or sodium chloride to selectively precipitate proteins?

Column based extraction

a method that employs selective binding of nucleic acid to a solid

matrix, such as silica that is packed in a column?

Magnetic bead-based extraction

a method that utilizes small particles with a paramagnetic core that

binds to nucleic acid?

Magnetic bead-based extraction

consist of an iron-oxide core covered by functional carboxylic group?

Chromatography-based extraction

(Ion exchange chromatography and Molecular exclusion chromatography)

DNA molecules and other simple and complex molecules in a complex matrix can be separated based on net molecular change or molecular size; method is based on typical chromatographic methods.

Chromatography-based extraction

(Ion exchange chromatography and Molecular exclusion chromatography)

This is used for crude cell lysates

EtBr-CsCl Gradient Centrifugation Method

The DNA sample is mixed with Cesium chloride, ultracentrifuged at 10,000 - 12,000 rpm for >10 hours. The DNA molecule separates based on density?

Cesium chloride

EtBr-CsCl Gradient Centrifugation Method

The DNA sample is mixed with _________, ultracentrifuged at 10,000 - 12,000 rpm for >10 hours. The DNA molecule separates based on density?

10,000 - 12,000 rpm for >10 hours

EtBr-CsCl Gradient Centrifugation Method

The DNA sample is mixed with Cesium chloride, ultracentrifuged at _______________. The DNA molecule separates based on density?

Cesium chloride

EtBr-CsCl Gradient Centrifugation Method

_____ allows interaction with negatively-charged DNA?

Ethidium bromide

EtBr-CsCl Gradient Centrifugation Method

Acts as an intercalating agent and allows supercoiled DNA to accumulate at lower densities

Ultraviolet light

EtBr-CsCl Gradient Centrifugation Method

The location of DNA can be easily visualized under?

Ethidium bromide

Cesium chloride

EtBr-CsCl Gradient Centrifugation Method

____________Are removed prior to precipitattion of DNA with?

EtBr-CsCl Gradient Centrifugation Method

This is used for plasmid-containing samples?

Alkaline extraction

The sample is suspended in an alkaline solution containing NaOH and SDS detergent, causing the denaturation of chromosomal DNA.

Potassium acetate

Alkaline extraction

______ added to neutralize the solution, causing chromosomal DNA to renature and precipitate from the solution.

Plasmid DNA

Alkaline extraction

_______ in the supernatant is isolated afrer centrifugation

Alkaline Extraction

This is used for extracting plasmid

NaOH

SDS detergent

In Alkaline extraction,

The sample is suspended in an alkaline solution containing __________ causing the denaturation of chromosomal DNA.

Cellulose-based paper

_______________-are impregnated with buffers, detergents and chelating agents that facilitate extraction of DNA

Chelex-100 Extraction

Chelex resin is a styrenedivinylbenzene material which chelates metal ions that act as cofactors for DNases.

Chelex resin

_________is a styrenedivinylbenzene material which chelates metal ions that act as cofactors for DNases.

Chelex

_______ prevents DNA from being digested by DNases during boiling, which is done to denature proteins and DNA

Chelex-100 Extraction

The resulting single-stranded DNA is concentrated from the supernatant after centrifugation.