modern research techniques 2 - welcome aboard ug3! · pdf filethe dna preparation can...

Modern Research Techniques

Total Marks 60• Final 20• Class Tests 10• Quizzes 5• Presentations 5• Lab 20

DNA ISOLATION• Deoxyribonucleic acid (DNA) isolation is an extraction process of DNA from various sources.

Methods used to isolate DNA are dependent on the source, age, and size of the sample. Despitethe wide variety of methods used, there are some similarities among them. In general, they aim toseparate DNA present in the nucleus of the cell from other cellular components.

• Scientists use DNA in a number of applications, such as - Introduction of DNA into cells and animals or plants - Diagnostic purposes. - Forensic science needs to recover DNA for identification of individuals• Presence of proteins, lipids, polysaccharides and some other organic or inorganic compounds in

the DNA preparation can interfere with DNA analysis methods, especially with polymerase chainreaction (PCR).

• Sources for DNA isolation are very diverse. Basically it can be isolated from any living or deadorganism. Common sources for DNA isolation include

- Whole blood - Hair - Sperm - Bones - Nails - Tissues, - Blood stains - Saliva - Buccal (cheek) swabs - Epithelial cells - Urine - Bacteria - Animal tissues, or plants

http://www.enotes.com/dna-isolation-methods-reference/dna-isolation-methods

Nature Protocols 1, 2320 - 2325 (2006)

Flow diagram to illustrate themajor steps of the modifiedCTAB (cetyltrimethylammoniumbromide) DNA isolation protocol

DNA is a polar molecule due to the negativecharges on it’s phosphate backbone, so it is verysoluble in water and less in phenol. This meansthat when the water(+DNA +protein) and phenolare mixed in the protocol, the DNA does notdissolve in the phenol, but remains in the waterphase.

Proteins are permanently denatured by the newsolvent environment provided by the phenol.

In water the polar residues on the outside of theproteins made them soluble in water, the phenol-induced folding changes forced the phenol-favoring residues to outside so that the proteinsare now more more soluble in phenol than inwater.

The phenol-soluble proteins partition to thephenol phase while, the water soluble, polar DNAmolecules stay in the water phase

Genomic DNA from solid biomaterials was directly isolated with anautomated DNA extractor, which was based on magnetic beadtechnology with a bore-mediated grinding (BMG) system. Themovement of the bore broke down the solid biomaterials, mixed crudelysates thoroughly with reagents to isolate the DNA, and carried thebeads to the next step. The BMG system was suitable for themechanical homogenization of the solid biomaterials and valid as anautomated system for purifying the DNA from the solid biomaterialswithout the need for pretreatment or disruption procedures prior to theapplication of the solid biomaterials.

Direct DNA isolation from solid biological sources withoutpretreatments with proteinase-K and/or homogenization throughautomated DNA extraction

Journal of Bioscience and Bioengineering Volume 103, Issue 3 2007 242 - 246

Overview of automated DNA extraction system (MX16). (A) MX16 system (W37×D31×H31 cm); (B) 16channels of BMG for parallel extraction; (C–E) BMG complex; (F) lateral view of BMG and cartridge;(G) apical view of prefilled cartridge; (H) bore; (I) schematic views of both bore and cartridge. A circlein panel B represents the BMG complex, which is composed of a magnetic bar and a bore tophysically extract genomic DNA and to move magnetic particles. Two-sided arrows in panel Brepresent a movement in a horizontal or vertical direction.


Direct DNA isolation from solid biological sources without pretreatments with proteinase-K and/or homogenizationthrough automated DNA extraction


DNA purification protocol usingBMG complex of MX16: (A)overview of prefilled catridge andapplicable samples; (B) DNAextraction procedure). In the firststep, a rod indicated as bore wasused for the mechanical disruptionof the solid samples in thepresence of chaotropic salts(compounds ability to disrupt thehydrogen bonds structure of DNA)and for the collection of themagnetic beads. The beadscarrying the DNA were transferredto subsequent chambers forwashing by repeated capture andrelease using a permanent magnet.Finally, the DNA carried by thebeads was released in the elutionbuffer.

Genomic DNA isolated from plant leaves (approximately 30 mg of soybean and citrus)using MX16 with different chaotropic reagents and mechanical extraction methods. (A)DNA purified using different lysis buffers: lane 1, TE; lane 2, NaClO4; lane 3, NaI; andlane 4, GuSN. (B) DNA extracted by three different lysing methods: no mechanicaltreatment (lanes 1, 4), vigorously vortexing for 10 min (lanes 2, 5), and BMG with MX-16(lanes 3, 6). The eluate (5 µl from 180 µl) of each sample was loaded onto a 1% agarosegel. Lane M, Lambda DNA HindIII marker.

Jang-Seu Ki , Ki Byum Chang , Hee June Roh , Bong Youb Lee , Joon Yong Yoon , Gi Young Jang



http://dx.doi.org/10.1263/jbb.103.242

Results of DNA isolation and PCR amplification. DNA was isolated from approximately 30 mg ofsoybean, onion, sweet corn, and citrus tissue, 30 mg of cow liver, short-necked clam and oyster, threeclippings of 5-mm mouse tail and 50 µl of pelletted bacterial cells. For forensic samples, DNA waspurified from one to three freshly collected hair roots and 100 µl of saliva and buccal swabs. PCRamplifications were carried out by following the standard protocol, as described in Materials andMethods. The eluate (5 µl from 180 µl) of each sample and 2 µl of PCR products were loaded onto 1%agarose gels. (A) Lane 1, Escherichia coli; lane 2, Streptomyces sp. type 1; lane 3, Streptomyces sp.type 2; lane 4, Streptomyces sp. type 3. (B) Lane 1, Bos taurus; lane 2, Mus musculus; lane 3,Ruditapes philippinarum; lane 4, Crassostrea gigas. (C) Lane 1, Glycine max; lane 2, Zea mays; lane 3,Citrus unshiu; lane 4, Allium cepa. (D) Lane 1, One hair root; lane 2, three hair roots; lane 3, buccalswab; lane 4, human saliva. Lane M1, Lamda DNA HindIII marker; lane M2, 100 bp A marker.



http://dx.doi.org/10.1263/jbb.103.242

Sensitive and automation friendlyIsolation of viral RNA/DNA using Dynabeads Saline

For high copy plasmid DNA:

Types Expected DNA YieldMini 2 - 20 µgMidi 20 - 100 µgMaxi 100-1,000 µgMega 1-5 mgGiga 5-20 mg

Low copy plasmid dna For low copy plasmid DNA:

Types Expected DNA YieldMini 0.1 - 10 µgMidi 10 - 50 µgMaxi 50 - 500 µgMega 0.5 - 2.5 mgGiga 2.5 -10 mg

http://www.acgtinc.com/dna_rna_extraction.htm

modern research techniques 2 - welcome aboard ug3! · pdf filethe dna preparation can...

Documents