MIDTERM LEC: Nucleic Acid Extraction Methods

It is to release the nucleic acid from the cell for use in subsequent procedures.

nucleic acid extraction

What are the two main types of nucleic acids

DNA, RNA

A molecules that carry genetic information in living things

Nucleic acids

purpose is to release nucleic acid from the cell for

• Detecting a specific pathogen

• Diagnosing disease and genetic disorders

Who is the person first Isolated the nuclein (DNA) from the WBCs he obtained from the pus on collected surgical bandages in a nearby hospital

Friedrich Miescher

Who is the person demonstrate semi- conservative replication of DNA

Meselson and Stahl

Although Miescher first isolated DNA from human cells in 1869 by

precipitation

the early routine laboratory procedures for DNA isolation were developed from

density-gradient centrifugation strategies

What are those Test Specimens Adequate for Analysis Without DNA Amplification

• blood

• Bone marrow

• Feces

• Mitochondria

What are those test that Specimens Adequate for Analysis With DNA Amplification

• bone teeth

• Hair follicles

• Fixed tissue

• Feces

Method used that Cell walls are not thick and can be lysed by high pH and detergents.

gram-negative bacteria

It is a surfactant that is often used in the process of cell lysis, or the breaking open of cells.

1% sodium dodecyl sulfate

Common treatment with detergent used

sodium dodecyl sulfat

In a solution, a strong base will react with water to produce Of what

0.2 M NaOH

In a solution, a strong base will react with water to produce Of what (0.2 M NaOH) in the presence of what

• Tris base

• EDTA

• glucose

Commercial reagents designed for isolation of DNA in amplification procedures, such as polymerase chain reaction (PCR), are used For what

yeast, filamentous fungi, and gram-positive bacteria.

It is held within free viruses or integrated into the host genome along with host DNA.

Viral DNA

What do you called immature viruses

Viroids

Nucleic acid in human blood or bone marrow comes mostly from What

White blood cells

Anticoagulants added to the collected sample will prevent clotting, which will trap the?

White blood cells

small vesicles (30 to 100 nm in diame- ter), which form by invagination and budding from the inside of cellular endosome vesicles and are secreted by living cells.

Exosomes

Exosomes contain nucleic acid and can be collected by what

Centrifugation

these sources of circulating nucleic acids can be detected for purposes of diagnostic and prognostic analyses. Such a test is called a

liquid biopsy

Dissociated by mincing the tissue

Fresh tissue

Disssociated by grinding and homogenizing the tissue

Frozen tissue

These purposes sources of circulating nucleic acids can be detected for purposes of diagnostic and prognostic analyses

Exosomes

May be deparaffinized by soaking in xylene and tissue is rehydrated by soaking it in decreasing concentrations of ethanol

Fixed, embedded tissue

Fixed, embedded tissue May be used directly without

dewaxing

What are the 3 types of tissue samples

• fresh tissue

• Frozen tissue

• Fixed, embedded tissue

What are the good tissue fixative influencing nucleic acid quality

• 10% buffered neutral formalin

• Acetone

It is the least damaging among tested fixatives, with mercury- based fixatives, such as Bouin’s and B-5, being the worst for DNA recovery

buffered formalin

this is accomplished using a combination of high salt, low pH, and an organic mixture of phenol and chloroform

Organic Isolation Methods

What buffer used for Organic Isolation Methods

Phenol & chloroform

Ethanol ratios

2:1

Isopropanol ratios

1:1

What are the two alcohol that used for molec- ular applications.

ethanol and isopropanol

What buffer used in Inorganic Isolation Methods

Sodium acetate

Does not include organic extraction step Which is phenol and chloroform

Inorganic Isolation Methods

Inorganic Isolation Methods in Sometimes called______, makes use of low-pH & high-salt conditions to selectively precipitate proteins, leaving the DNA in solution

salting out

Uses solid matrices (silica-based products) to bind & hold DNA for washing

Solid-Phase Isolation

What another chemical used to remove silica and then it transfer to another tube

Elute DNA

In solid phase isolation, What products were shown to effectively bind DNA in high-salt conditions.

Silica-based

Preparation of samples for isolation of DNA on solid-phase media starts with

Cell lysis

It is dewaxed with xylene or other agents

Paraffin embedded specimen

What method used in Hair with root is incubated (95C) for 10 mins in NAOH buffer

Simple alkaline lysis method

What method that Hair sample can be incubated (56C) for 2h with buffer containing Tris-HCl, EDTA, NaCl, SDS, DTT, & proteinase K, followed by gentle mixing & incubation (60C) for 2h or until the hair has dissolved completely

Smooth chemical digestion method using Dithiothreitol (DTT)

Suspended in lysis buffer which includes Tris, EDTA, SDS, & proteinase K

Buccal swabs

Buccal swab is incubated in and what hours

58 degree Celsius, 1-3 hours

What uses for buccal swabs

Cotton swabs

This Samples from saline rinses need to be processed/ frozen immediately after collection

Mouthwash method

Inverted & swirled in a specimen cup to create a homogenous suspension of cells followed by the centrifugation

Urine specimen

Urine specimen Is incubated

56 degree celsius for 2 hours

What method used for genomic DNA purification from saliva

• buccal swab

• Mouthwash method

What other application for puposes is to release nucleic acid from the cell

• Forensics

• Paternity tests

• Ancestry tracking

• Genetic engineering

• Vaccines

• Hormones