1. Pre-treatment and Washing 2. Cell lysis 3. Removal of contaminants 4. DNA Precipitation 5. DNA Resuspension

Nucleic Acid Extraction Flashcards by Kyra Catedral

This is a preliminary step for various applications, including research, medical diagnostics, and forensic investigations.

NA extraction

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The extracted nucleic acid should be free from contaminants, such as (3)to ensure accuracy in downstream applications.

proteins, carbohydrates, and lipids,

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– Found in enveloped viruses, these proteins help in host cell recognition and attachment.
– A lipid membrane surrounding some viruses, derived from the host cell membrane.
– The nucleic acid (DNA or RNA) that carries the genetic information.
– A protein coat that protects the viral genome.
– Found in some viruses, it contains proteins that assist in viral replication and immune evasion.

• Envelope Protein
• Envelope
• Viral Genome
• Nucleocapsid
• Viral Tegument

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CHOOSING A NUCLEIC ACID EXTRACTION METHOD

Selecting the appropriate extraction technique is crucial and depends on several factors:

Efficiency
Sufficiency
Purity

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• The method should be able to effectively release and isolate nucleic acids with minimal loss.
• It should also be affordable and practical, especially for routine laboratory use.

Efficiency of Extraction & Cost-Effectiveness

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• The extracted DNA or RNA must be in a sufficient amount to be used in further analyses like PCR, sequencing, or hybridization techniques.
• Low yields may lead to unreliable results

Sufficiency of Quantity for Downstream Applications

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The final extract must be free from contaminants to avoid interference in downstream applications.

Purity of the Final Nucleic Acid Extract

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The process involves three major steps:
– Separating nucleic acids from other cellular components.
– Removing proteins, carbohydrates, lipids, and other impurities.
– Adjusting the nucleic acid quantity for specific applications.

• Isolation

•	Purification

•	Concentration

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The extracted nucleic acids are essential for various fields:

Scientific research
Medical
Forensic

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• Used in molecular biology to study gene expression, mutations, and genetic variation.
• Helps in biotechnology applications, such as genetic engineering, cloning, and gene therapy.

Scientific Research

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• Identifying the etiology of infections – Helps detect viral, bacterial, fungal, or parasitic infections through PCR, RT-PCR, or sequencing.

• Determining possible drug resistance – Identifies genetic mutations linked to antimicrobial resistance, such as in Mycobacterium tuberculosis or HIV.

• Predicting disease progression – Genetic markers help assess the risk and prognosis of diseases like cancer and inherited disorders.

Medical Applications

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Forensic Applications

– DNA extraction is used to establish biological relationships through short tandem repeat (STR) analysis.

– DNA fingerprinting helps in criminal investigations by matching evidence DNA with suspect profiles.

• Paternity testing

•	Identification of suspects

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DNA extraction

Pre-treatment and Washing
Cell lysis
Removal of contaminants
DNA Precipitation
DNA Resuspension

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Before actual DNA extraction, some biological samples require_____ to break down barriers that may hinder efficient DNA isolation.

pre-treatment

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PRE-TREATMENT AND WASHING• Example: Formalin-fixed paraffin-embedded (FFPE) tissue samples require______ to remove the wax before DNA isolation.
• ______of samples allows for the calculation of the final DNA yield percentage, which is important in research and diagnostics to assess extraction efficiency.

deparaffinization

Weighing

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The______ step disrupts the cell membrane and nuclear envelope to release intracellular contents, including DNA.

cell lysis

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Cell lysis can be…

Physical
Solution-based

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Physical Lysis (GSBF)

Mechanical methods are used to physically break apart cells:
________– Sample is manually or mechanically crushed (e.g., with a mortar and pestle in liquid nitrogen).
_________– High.. forces break open cells, commonly used for large-scale bacterial cultures.
_________–… are agitated at high speed to disrupt cells, effective for tough cell walls (e.g., yeast and fungi).
_________– Repeated freezing and thawing create ice crystals that rupture cell membranes.

Grinding
Shearing
Bead Beating
Freeze-Thaw Cycles

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Benefits:

• Highly effective for a wide range of cell types.

• Allows control over buffer composition to remove contaminants.

• Ensures a high level of lysis efficiency.

Physical lysis

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Disadvantages:
• Requires specialized equipment.
• Not always reproducible due to variations in mechanical force.
• Can cause localized heating, leading to protein denaturation and degradation of cellular components.

Physical lysis

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Solution-Based Lysis

This method uses (2) to break down the cell membrane and nuclear envelope.

chemical agents and enzymes

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Chemical Lysis Agents: ADC

__________– Disrupts lipid membranes and denatures proteins.
__________– Breaks apart lipid bilayers and helps dissolve proteins.
__________– Chelates divalent ions like Mg²⁺, which are required for nucleases that degrade DNA.

• Alkaline Solutions (NaOH)
• Detergents (SDS, CTAB)
• Chaotropic Agents (EDTA)

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Enzymatic Lysis Agents: PLL
_______– A broad-spectrum enzyme that degrades proteins, including nucleases that may degrade DNA.
_______– Breaks down bacterial cell walls by targeting peptidoglycan.
_______– Helps dissolve lipid-rich cell membranes.

• Proteinase K
• Lysozyme
• Lipase

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Benefits:
• Rapid, gentle, and efficient method.
• Works well for small sample volumes and high-throughput extraction.
• Can be combined with mechanical methods for enhanced cell disruption.

Solution based lysis

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Disadvantages: • ***Some buffer components need removal*** before DNA analysis. • ***High concentrations of salts or detergents can interfere with protein assays and mass spectrometry.*** • Works best with cultured cells, but may be ***less effective for solid tissues.***

Solution based lysis

After lysis, the sample contains DNA mixed with proteins, RNA, lipids, and cellular debris. Those must be removed to ensure high-purity DNA extraction.

REMOVAL OF CONTAMINANTS

REMOVAL OF CONTAMINANTS Protein Removal ______– Break down and digest proteins that could interfere with DNA extraction. ***Can also degrade nucleases*** _______– An ***anionic detergent*** that denatures proteins and ***makes them negatively charged*** for easier removal.

• Proteases (e.g., Proteinase K) • Sodium dodecyl sulfate (SDS)

REMOVAL OF CONTAMINANTS RNA Removal ________ selectively degrade RNA, leaving only DNA in the sample. • This step is critical when extracting DNA for PCR or sequencing, as RNA contamination can lead to erroneous results.

RNase enzymes

(Separation of DNA from Solution)

DNA PRECIPITATION

Once contaminants are removed, DNA must be separated and collected from the solution. The choice of precipitation method depends on the type of extraction used.

DNA PRECIPITATION

DNA PRECIPITATION Materials (3)

Organic Inorganic Solid phase

• Uses ***high salt concentrations*** in a low pH environment along with a ***1:1 mixture of phenol and chloroform.***

Organic Extraction (Phenol-Chloroform Method)

• Forms a ***biphasic emulsion*** after centrifugation. • ***DNA remains in the aqueous layer, *** while proteins and other impurities partition into the organic phase. • DNA is then ***precipitated using cold ethanol*** in a low-temperature environment.

Organic Extraction (Phenol-Chloroform Method)

Organic Extraction • Uses high salt concentrations in a low pH environment along with a 1:1 mixture of_______ • Forms a______ after centrifugation. • DNA remains in the aqueous layer, while proteins and other impurities partition into the organic phase. • DNA is then precipitated using_____ in a low-temperature environment.

phenol and chloroform. biphasic emulsion cold ethanol

Cluster of solvent molecules surrounding and attaching to solute molecules in solution

Solvation shell

DNA PRECIPITATION Advantages: • Highly efficient and reliable. • Suitable for a wide range of sample types.

ORGANIC

DNA PRECIPITATION Disadvantages: • Time-consuming due to multiple tube transfers. • Uses toxic reagents (phenol, chloroform).

ORGANIC

(“Salting Out”)

Inorganic Extraction

• Uses low pH, high salt conditions to selectively precipitate proteins while keeping DNA in solution. • DNA is then precipitated using isopropanol.

Inorganic Extraction (“Salting Out”)

Inorganic Extraction (“Salting Out”) • Uses low pH, high salt conditions to selectively precipitate proteins while keeping DNA in solution. • DNA is then precipitated using______.

isopropanol

DNA PRECIPITATION Advantages: • Faster and safer than organic methods. • Uses non-toxic chemicals. • Fewer steps, reducing contamination risks.

Inorganic Extraction (“Salting Out”)

DNA PRECIPITATION Disadvantages: • Incomplete removal of salts may cause band shifting during gel electrophoresis, making DNA appear in the wrong position.

Inorganic Extraction (“Salting Out”)

(Column-Based Methods)

Solid-Phase Extraction

• DNA binds to a solid matrix (e.g., silica, magnetic beads, anion exchange resins). • After several washing steps, DNA is eluted from the column in a purified form.

Solid-Phase Extraction

SOLID PHASE • DNA binds to a solid matrix (e.g.,_____). • After several washing steps, DNA is eluted from the column in a purified form.

silica, magnetic beads, anion exchange resins

DNA PRECIPITATION Disadvantages: • Expensive due to the cost of commercial kits and specialized columns.

SOLID PHASE

DNA PRECIPITATION Advantages: • Fast and highly efficient method. • Produces high-quality DNA. • Commonly used in commercial DNA extraction kits.

SOLID PHASE

After precipitation and purification, the extracted DNA must be ______ in a buffer for long-term storage and further analysis.

DNA RESUSPENSION (Storage and Stabilization of DNA)

DNA RESUSPENSION maintains a basic pH, which prevents interactions between DNA, histones, and polycationic amines. Used to prevent DNA degradation by DNases.

Tris-EDTA (TE) Buffer

Resuspension in_____is done so the extract can be stored and diluted for downstream uses It chelates divalent ions that could potentiallyfo m slats with the phosphate groups of DNA and inhibits DNAses

Tris-EDTA (TE) Buffer

DNA RESUSPENSION Storage Conditions • Short-term: • Long-term:

4°C (for a few days to weeks) –20°C to –80°C (to preserve DNA integrity)

RNA is significantly less stable than DNA because it has a_______ on the ribose sugar, which makes it more prone to______.

2′ hydroxyl (-OH) group hydrolysis

• _______are enzymes that degrade RNA and are ubiquitous in the environment (found on human skin, lab surfaces, and even in the air). They are highly resistant to denaturation and can remain active even after autoclaving or exposure to extreme conditions.

RNases (ribonucleases)

• Freezing the sample in______ or immersing it in an______ immediately after collection prevents RNA degradation before extraction. • Lab equipment, reagents, and workspaces must be RNase-free (e.g., using DEPC-treated water and RNase inhibitors).

liquid nitrogen RNase-inactivating buffer

METHODS OF RNA EXTRACTION

1. ORGANIC RNA EXTRACTION 2. SOLID OHASE EXTRACTION

RNA EXTRACTION This method involves using _____ solvents to extract RNA while effectively removing DNA and proteins. It is a widely used method due to its efficiency in stabilizing and protecting RNA.

ORGANIC RNA EXTRACTION (PHENOL-BASED METHOD)

ORGANIC RNA EXTRACTION Cell Lysis • Cells are lysed in a_______ or ________ to denature proteins and prevent RNase activity. • _______may be added to further protect RNA from degradation. •______ treatment may also be included in this step to remove contaminating DNA.

detergent-based solution or phenol with high salt concentrations (0.2–0.5 M NaCl) RNase inhibitors DNase

ORGANIC RNA EXTRACTION Protein Removal Two methods are commonly used for protein separation:

Option 1: Acid Phenol: Chloroform: Isoamyl Alcohol Extraction Option 2: TRIzol Method (Acid-Guanidinium-Phenol-Based Extraction)

RNA is precipitated using... • ______ or ______volume is added to the RNA-containing aqueous phase. • The solution is incubated at -20°C to -80°C to enhance RNA precipitation. • RNA is pelleted by centrifugation and washed with cold ethanol to remove impurities.

2× ethanol 1× isopropanol

RNA Resuspension • The RNA pellet is resuspended in ______to ensure that TNA will not be degraded while being stored or during use

nuclease-free water

ADVANTAGES • Rapid elimination of nucleases • Stabilization of RNA • Can be used for smaller or larger samples • Protocols are well-established and more routinely used DISADVATAGES • Time-consuming • Laborious • Makes use of hazardous reagents

ORGANIC RNA EXTRACTION

Lysate is added to the spin column (microfuge tubes) with high salt chaotropic buffer Cell lysis is similar to organic extraction RNA is washed and eluted off of the membrane

SOLID PHASE RNA EXTRACTION

ADVANTAGES • Simple and straightforward • Used in kits, making them highly convenient • Usable in large-scale extractions and automated methods DISADVANTAGES • Large amounts of sample cannot benused in one go as it can clog the membranes • Expensive

SOLID PHASE RNA EXTRACTION

WHY ASSESS NUCLEIC ACID QUANTITY AND QUALITY?

• To check the concentration and purity of yield • Determines success of isolation • Calculations used for downstream application

• Knowing the concentration allows for accurate dilution calculations for further analysis. • Impurities such as proteins, phenols, and salts can interfere with enzymatic reactions and distort experimental results.

To determine the concentration and purity of the extracted nucleic acid

• A high yield of DNA or RNA with minimal contamination confirms that the extraction process was efficient. • Poor-quality extractions may contain RNA degradation, protein contamination, or salt carryover, which can affect results.

To assess the success of the isolation process

• Many molecular biology experiments require precise nucleic acid amounts for optimal performance. • Example: In qPCR, an inaccurate input of nucleic acid concentration can lead to unreliable quantification of gene expression.

To ensure accurate calculations for downstream applications

METHODS USED FOR NUCLEIC ACID QUANTIFICATION AND PURITY ASSESSMENT

1. Spectrophotometry (Absorbance-based method) 2. Agarose Gel Electrophoresis (Integrity assessment)

SPECTROPHOTOMETRY Principle:

Beer-Lambert’s Law

states that the absorbance (A) of a substance is ***directly proportional*** to its concentration (C), the molar extinction coefficient (ε), and the path length (L) of the light passing through the sample

Beer-Lambert’s Law

• 1 absorbance unit (A260) =DNA • 1 absorbance unit (A260) =RNA

50 μg/mL of double-stranded DNA (dsDNA) 40 μg/mL of RNA

PURITY ASSESSMENT OF NUCLEIC ACIDS Determined by…

UV spectrophotometry

Ratio often used to assess purity

A260/A280 Ratio

• A260/A280 ratio often used to assess purity o Absorbance at_____ for nucleic acids o Absorbance at_____ for proteins o Additional_____ for other contaminants and 270 nm for phenols

260 nm 280 nm A230 nm

(2) absorb light at 260 nm, while____ absorb light at 280 nm (due to aromatic amino acids like tryptophan and tyrosine).

DNA and RNA proteins

A260/A280 Ratio (Protein Contamination) • An A260/A280 ratio of_____indicates a pure nucleic acid sample. • Ratios_____ suggest protein contamination (residual proteins from the extraction process). • Ratios _____may indicate RNA contamination in a DNA sample.

~1.8–2.0 <1.8 >2.0

Contaminants like phenols, carbohydrates, and chaotropic salts absorb at_____.

230 nm

A260/A230 Ratio (Salt, Carbohydrate, and Phenol Contamination) • An A260/A230 ratio of_____ is ideal. • Ratios____ indicate contamination, which may affect downstream enzymatic reactions.

2.0–2.2 <2.0

• Requires only 1-2 uL of sample instead of needing 50-75 uL • Displays the entire absorbance spectrum in graphical form • Can determine a wide range of sample concentrations without serial dilution (2 ng-15,000 ng/uL)

Nanodrop spectrometry

Nanodrop spectrometry • Requires only ____of sample instead of needing____ • Displays the entire absorbance spectrum in graphical form • Can determine a wide range of sample concentrations without serial dilution (2 ng-15,000 ng/uL)

1-2 uL / 50-75 uL

• Separation method based on size under the influence of an electric current (max: 15 V) • A calibration curve is done after to plot the log of the observed molecular weight based on the marker against the distance traveled by each of the bands • After running, the bands of interest can be isolated out and reextracted for further studies

AGAROSE GEL ELECTROPHORESIS

• 1 absorbance unit (A260) =DNA • 1 absorbance unit (A260) =RNA

50 μg/mL of double-stranded DNA (dsDNA) 40 μg/mL of RNA