Research Methods 1.1 Nucleic Acid Extraction

cloning

production of multiple exact copies of a piece DNA in order to produce enough DNA for further study. this is done through isolating the DNA that encodes the gene of interest from the organism of study

DNA extraction objective

separate the DNA from other cellular compones and to remove contaminants from the DNA while the DNA remains intact

gDNA extraction disrupts

plant cell walls and outer cell membranes along with eukaryotic cells with membranes around nucleus and organlles because most of the genes are contained in genomic DNA located in the nucleus

basic steps of DNA extraction

1. grow or collect cells

2. disrupt cell walls and membranes by physical means

3. lyse cell membranes by chemical means

4. remove cellular debris

5. remove proteins and purify DNA

6. concentrate DNA

plant structure

multicellular eukaryotic organisms distinguished by rigid cell walls for shape and support and chloroplasts for photosynthesis

why must cell walls be disrupted

in addition to lysing cell membranes, cell walls must be disrupted because they have multiple layers of cellulose and polysaccharides that colocalize with DNA making it more difficult to separate from other components

why is extraction done

to minimize potential DNA damage from nucleases digesting DNA and easily disrupted organlles like plant vacuoles with acidic contenet that can damage DNA

polyphenols

some plants can also have polyphenols that when lysed without special precautions can irreversibly bind to DNA making it useless for subsequent experiments

what is the main difference between gDNA and organelle DNA

the size of the DNA, very long DNA is isolated from gDNA because the GAPC gene is encoded in gDNA

harvesting of cells

harvest fresh plant tissue, young leaves are the best source for gDNA because leaves that are still growuing have a greaterreation of cytoplasmic volume and fewer chemicals that interfere with DNA isolation, also fresher samples will have higher yields of DNA

grind cells

physically disrupt plant cell tissues, due to rigid cell walls plant tissue must be physically ground or crushed prior to DNA isolation through the use of a mortar and pestle and this is done in the presence of a lysis buffer to prevent DNA damage by cell contents. the plant cells must be broken up into fine small pieces to make sure the lysis buffer can access it with detergent since it breaks up the membranes as it is a phospholipid.

lyse cells

lysing of cells in solutions containing enzymes and chemicals that disrupt cell membranes, components include a chelating agent, buffer, detergent, chaotropic salts, reducing agent, and PVP

removal of cellular debris

removing insoluble components sich as remains of the cell wall and membranes by centrifugation leaving DNA in the supernatant

purify DNA

remove contaminating proteins which will be in the supernatant with the DNA by using silica column purification

concentrate DNA

elute purified DNA from silical column with a small volume of elution buffer (water)

chelating agent

EDTA remove Mg2+ by binding to it and thus inhibits nucleases that could digest DNA as well as destabilizing the cell wall and cell membrane, it degrades and inactivates enzymes

buffer

lysis buffer must have buffering capacity. when cells are lysed, acidic compounds are released from vacuoles so the lysis buffer must be formulated to ensre that the lysate does not change its pH dramatically and this is done through Tris with a pH of 8.0.

detergent

cell membrane needs to be disrupted which can be done by detergents since they break up cell membranes by removes lipid molecules from the membranes. detergents include SDS, but nonionic detergents can also be used such as X-100 or CTAB since they are milder than ionic detergens and leave the proteins intact and functional

chaotropic salts

disrupt proteins and membranes by destabilizing their 3D structure. guanidine thiocyanate helps to denature proteins and cell membranes and it works by disrupting the hydrogen binding between water molecules reducing the stability of the native state of proteins and reducing them. guanidine thiocyanate also allows DNA to bind to a silica column and isolate DNA.

reducing agent

DTT minimizes protein oxidation caused by covalent modification of proteins by reactive oxygen species or oxidative stress. oxidative stress can destroy DNA such as the presence of polyphenols coming into contact with oxygen during the extraction process and causing oxidative stress

PVP

works with high salt concentration and binds polyphenols preventing them from complexing with DNA. high salt concentration prevents coprecipitation of polysaccharides with the DNA.

what can vortexing lysate do

break dna and cause shearing

how does DNA purification work

removal of contaminants is done by silica based purification which relies on the strong binding od DNA to silica in the presence of high salt concentration like guanidine thiocyanate which disrupt hydrophobic interactions. binding can be due to exposure of anions on DNA and silica as a result of dehydration by the salts and phonphates on the DNA bind to silica through formation of a cation bridge formed by the salt. DNA can then be released from silica by reducing salt concentration.

how does DNA concentration work

washing DNA with 70% ethanol helps to bind DNA to the column since the salts that help DNA stick to the column can in the solution at this concentration. if the ethanol concentration would be 100% there would be no dissolving and at a concentration less than 70%, DNA loss would occur with each wash. In general, washing helps purify the DNA to remove remaining protines, contaminants, polysaccharides and lipids. to elute DNA, sterile warm water is used and warming it helps detachment while a small amount of water still keeps the DNA concentrated. in the final sample, gDNA and RNA is present.

importance of ethanol

70% ethanol helps DNA bind to the column, ethanol is added to the supernatant in order to bind DNA to the column and since it is less hydrophilic to water DNA aggregates. the 70% concentration works for washing the contaminants hydrophillically and does not wash of the DNA as it is hydrophobic.