DNA Laboratory Methods Flashcards
Why is it necessary to culture cells?
In order to understand how cells function- in normal situations and in specific scenarios
What cellular functions do we want to understand with cell culture? (2)
- Growth characteristics
- Roles and structures of different proteins, which may dictate cellular functions
Why is it necessary to study nucleic acids?
Protein comes from RNA, so studying RNA gives us an idea of gene expression. Studying DNA allows us to learn about gene identification and gene function
Key techniques for analyzing and manipulating DNA (6)
- DNA cleavage by restriction enzymes- creates pieces of DNA
- DNA ligation- needed to create recombinant DNA
- DNA cloning
- Nucleic acid hybridization & sequence detection
- Rapid DNA sequencing
- Monitoring of gene expression
What sources is DNA extracted from
You can either grow cells or remove tissue from an animal to extract DNA
Steps of DNA extraction (4)
- Cell lysis- breaking the cells open
- Protein digestion (optional)- you aren’t interested in proteins if you are extracting DNA
- RNA digestion- only want to isolate DNA
- Need to purify DNA away from digested materials, detergents, etc
Cell lysis
Breaking the cells open in order to get the DNA out. This may be done through grinding, physical disruption (passing through syringe), sonication, detergents. Chaotropic buffers are very important here as well
Proteases
Used for protein digestion
RNases
Used for RNA digestion
Methods for DNA purification (3)
- Alcohol precipitation
- Phenol-chloroform extraction
- Minicolumn purification
Alcohol precipitation
Uses ice-cold ethanol or propanol. DNA is insoluble in alcohol, so the cold alcohol will cause the DNA to pellet (precipitate out) in the centrifuge. Improved by increasing ionic strength (sodium acetate)
Phenol-chloroform extraction
Phenol denatures proteins, so you may not want to use this method in all cases. Use a centrifuge so denatured proteins will stay in the organic phase- the aqueous phase contains DNA. The aqueous phase is then mixed with chloroform to remove phenol residues
Minicolumn purification
Most popular and efficient method of DNA purification, which is often combined with alcohol precipitation. Still uses the principle of DNA pelleting in alcohol. Has a lysis buffer which contains chaotropic salts, detergents, and proteinase K (digests proteins).
Purpose of detergents and proteinase K in a lysis buffer
Aids in the lysis and solubilization of lipids and proteins. Proteinase K works best on denatured proteins to digest them
Examples of chaotropic salts
HCl, guanidine thiocyanate, urea, & lithium perchlorate. These salts have chaotropic activity
Chaotropic activity
Chaotropic salts destabilize bonds. Specifically includes hydrophobic interactions, van der Waals interactions, and hydrogen bonds which are destabilized. The proteins are then destabilized/denatured. We also add in magnesium and calcium chelators to prevent DNases from functioning- we don’t want anything to destroy the DNA
Steps of minicolumn purification (4)
- Lysate for the lysis buffer is run through a silica column
- Column wash with chaotropic salt and ethanol. The ethanol wash is especially important because it precipitates the DNA and causes it to stick to the silica membrane
- Column dry to evaporate all of the ethanol
- Elution of the DNA- usually with TE (Tris-EDTA) buffer (pH 8-9) as DNA is more stable at a high pH. Although water may be used, TE is better for larger DNA
Binding/spin column
Used for DNA extraction via minicolumn. The binding matrix is composed of silica. Also contains a lysis buffer that is critical for DNA binding- the buffer disrupts hydrogen bonds which disrupts association of DNA with water and promotes binding to the silica. An alcohol (like ethanol or propanol) is usually added for precipitation. DNA interaction with water is disrupted, so it precipitates and influences DNA binding to silica
How is DNA quantified after minicolumn purification?
Uses a spectrophotometer to measure the absorbance at 260 nm, which is the absorbance of DNA. We typically take a ratio of the absorbance at 260 nm to the absorbance at 280 nm, which is the absorbance of proteins. Therefore, the 260:280 ratio is the ratio of DNA to proteins. Purified DNA should have minimal proteins. 1.8 is considered reasonably pure, anything less has too much protein and purification was not successful
What is the function of plasmid prep?
When extracting DNA from prokaryotic cells, you have to consider that they have extrachromosomal DNA (plasmids). You must separate plasmid DNA from genomic DNA- this is done based on size, as genomic DNA is much bigger
Steps for plasmid prep (3)
- Alkaline lysis- the lysis buffer has NaOH, which completely denatures all DNA
- Sample neutralization- DNA reanneals. Smaller plasmid DNA reanneals more quickly than genomic DNA
- Plasmid DNA is purified via the minicolumn- genomic DNA is pelleted prior
Restriction enzymes
Once DNA has been extracted, you have long strands. Restriction enzymes are used to isolate specific pieces of DNA. They recognize specific DNA sequences to produce DNA fragments of strictly-defined size. Sequences are usually 6 base pairs long and palindromic. May be used to create recombinant DNA
Blunt ends
When restriction enzymes cleave right through a strand of DNA
Staggered ends
When restriction enzymes produce cohesive “sticky” ends- single stranded overhangs, which are used to make recombinant DNA. The single stranded overhangs will base pair with each other
Recombinant DNA
When DNA is spliced together to create DNA that is not found in nature. Restriction enzymes may be used to create it.
DNA gel electrophoresis
DNA is loaded into a slab gel (a matrix through which DNA is propelled) and the DNA is run from negative to positive. As DNA has a uniform negative charge, it is propelled along the gel to the positive end via an electrical current. The DNA is separated based on size- the smaller pieces of DNA will travel farther, as the larger pieces have a harder time traveling through the matrix. It appears as bands that must be visualized
Polyacrylamide gel
Used to conduct polyacrylamide gel electrophoresis (PAGE). It is great at separating DNA fragments that are less than 500 base pairs long, and is able to resolve size by a difference of one base pair. Really only necessary if we need fine separation of DNA fragments
Agarose gel
A gel that is much more porous and dilute. It results in better separation of larger pieces of DNA