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
Methods to visualize DNA bands after gel electrophoresis (2)
- Ethidium bromide (EtBr)
- Radiolabeling
Ethidium bromide (EtBr)
One method used to visualize bands after DNA gel electrophoresis. It is added to the gel or buffer and fluoresces under UV excitation. Binds to and labels all DNA in one sample, indicating the location of all DNA bands. DNA hybridization probes are needed to detect specific DNA sequences, since EtBr detects ALL sequences
Radiolabeling
One method used to visualize DNA bands after DNA gel electrophoresis. Incorporates radioisotope (P32) into DNA prior to electrophoresis. This method is much more sensitive but also more dangerous, not used frequently
DNA hybridization probes
Single stranded DNA probes used to detect specific DNA sequences after gel electrophoresis. The probes are created through DNA labeling, which involves incorporation of radioactive nucleotides into existing DNA. Now, we tend to use fluorescent nucleotides for safety reasons
How is DNA hybridization conducted? (2 steps)
- DNA is first denatured (heated to 100 degrees Celsius)
- The probes will then hybridize (or anneal) if kept at 65 degrees Celsius following denaturation
Uses of DNA hybridization
This method is very sensitive and can detect as low as 1 molecule of a complementary sequence. May be used to determine how many copies of a sequence is present. Can also be used to find genes of interest or to detect RNA
Stringent hybridization
When the annealing temperature is kept close to the ideal temperature for the specific gene. The probe will then only hybridize with a perfect match (the identical sequence)
Non-stringent hybridization
When the annealing temperature is lower, it promotes hybridization of imperfectly paired DNA. This is useful for detecting related, yet non-identical genes
Applications of DNA hybridization
- Southern blotting
- Northern blotting
- Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization (FISH)
Direct hybridization of fluorescent probes on chromosomes or in intact cells (not in a gel). The fluorescent probes can detect parts of the chromosome with a high degree of sequence similarity in a portion of the tissue (in situ). The probes can be single stranded DNA or RNA (riboprobes). Can detect specific sequences in either DNA or RNA. Requires microscopy.
Southern blotting
Gel electrophoresis technique used to detect DNA
Northern blotting
Gel electrophoresis technique used to detect RNA
How are southern and northern blotting conducted? (6)
- DNA is digested (not for RNA) and run on gel
- DNA is then denatured by NaOH (not needed for RNA)
- DNA or RNA are then blotted to nitrocellulose or nylon by capillary action or a vacuum
- Membrane is blocked with non-specific probes (non-specific DNA)
- Incubation with specific probes
- Develop- can we detect fluorescence?
Tags used in FISH
Different tags can be used- fluorophores, tags detectable by antibodies, or biotin. Biotin is advantageous because streptavidin will ramp up the fluorescence signal
Nick translation
Makes slightly different probes for FISH. Single-stranded nicks are created in DNA and sealed by DNA Pol using the labeled nucleotides. Only some nucleotides are fluorescently labeled, rather than all of them
How is FISH conducted? (4)
- May be done on fixed cells or chromosome preps- Interphase or metaphase chromosomes firmly attached to substrate (usually glass)
- Repetitive DNA sequences must be blocked with nonspecific short DNA fragments
- Probes applied & incubated for ~12 h
- Entire chromosomes are probed- can’t do that with Southern blot
Applications of FISH (4)
- Identification/prediction of developmental disabilities- use FISH to detect sequences in parents’ and child’s DNA, assess risk of future disabilities
- Cancer diagnosis, assessment of remission
- Species identification
- Comparative genomic hybridization
How is FISH used in species identification?
It is used to diagnose infection with fastidious organisms, as well as in microbial ecology
How is FISH used in comparative genomic hybridization?
Compares hybridization strength to detect major disruptions in duplication process of DNA
What is required for PCR?
DNA Pol, nucleotides (dNTPs), primers, different temperatures (cycles) to separate the DNA. Replication fork machinery like helicase, primase, SSB proteins, and DNA ligase are NOT required
Phases of PCR (3)
Melting (94-96 degrees C) to separate the DNA strands, annealing (varies ~55-65 degrees C), elongation (~72 degrees C) where DNA polymerase begins synthesis
PCR gene amplification
Taq polymerase is the enzyme needed for this process. Uses a primer for the beginning of the gene complementary to the top strand, and a primer for the end of the gene that is complementary to the bottom strand. Initially you may get some segments that are longer than your gene of interest. As the cycles go on, you will get many more copies of the specific gene. PCR is extremely sensitive, it can detect a single DNA molecule in a sample. PCR has mostly replaced hybridization techniques like southern blots to identify specific DNA in samples
Importance of PCR (6)
- Primers are highly specific
- Detect specific genes in specific organisms
- Confirm that a specific gene is missing
- Use to assess infection- amplifies 16S rRNA from prokaryotes
- Integral in forensics
- Invaluable to genetic and molecular cloning
How is PCR used in genetic and molecular cloning?
Used to create mutated or recombinant genes, or to knockout genes
DNA fingerprinting
PCR is part of this process in forensics. Hypervariable microsatellite or variable number of tandem repeat (VNTR) DNA sequences are amplified. Individuals inherit different variant of VNTR sequences from mom and dad (1 from each). Two unrelated individuals will not contain the same pair of sequences, so you can use the DNA found at a crime scene to narrow down who could have committed the crime
PCR to detect RNA
Complementary DNA (cDNA, which does not contain introns) is created from RNA using reverse transcriptase- this is called RT-PCR. All DNA must be degraded first, then cDNA is subjected to the conventional PCR method
Dideoxy method
One method of DNA sequencing that uses dideoxyribonucleoside triphosphates. These nucleosides lack an extra 3’ OH, so the DNA chain is unable to extend beyond this nucleotide. DNA polymerase requires the 3’ OH group
How is dideoxy DNA sequencing conducted?
In one example, the dideoxy method will be used so a set of DNAs of different lengths will be produced, with each terminating at different adenine nucleotides. The exact lengths of the DNA fragments are then used to precisely determine the position of each A in the chain. The DNA products can then be run on a gel so they will be separated based on size. Each of the 4 dideoxynucleotides can be used in 4 separate reactions in order to determine the full DNA sequence
Next-generation sequencing (NGS)
A method of DNA sequencing that uses the same dideoxy method but is completely automated. Different-colored fluorescent dyes are used on each dideoxy ribonucleotide in order to distinguish between them. Run in same tube on single lane of a gel & read by computer
Methods of studying gene function
- Delete the gene- knockout
- Silence expression of the gene- knockdown
- Mutate the gene- mutant
- Look for phenotype- appearance or behavior
Methods of creating gene knockouts (2)
- Homologous recombination in yeast- yeast have intricate recombination machinery and are primed for recombination. This is complicated because genes must be introduced into the yeast if they are not originally yeast genes
- Other organisms- make use of transposons, which can be random and frustrating
Recombineering
An improved method of studying gene knockouts, making use of elements similar to the yeast system. Includes the lambda red system and gene cutting, such as CRISPR-Cas 9
Lambda red system
Homologous recombination aided by lambda red recombinase/integrase. Utilized mainly for gene knockouts in bacterial cells
RNA interference (RNAi)
Small interfering RNAs base pair with mRNA and destroy it. Silences mRNA transcripts- knockdown
Transformation methods (3)
Non-chemical
1. Heat-shock- briefly raise the temperature to transiently make the membrane more permeable and introduce knockout machinery
2. Electroporation- electricity
3. Sonoporation- ultrasonic waves
Nucleofection
A method of electroporation that uses a specific voltage and reagents that allow for permeation of the nuclear membrane
Transfection methods (3)
Chemical/vector
1. Liposome transfection
2. Viral envelope transfection
3. Viral particles
Liposome transfection
Liposomes are artificial vesicles. Foreign objects are put into the vesicles in the hopes that they will fuse with or be taken up by the cell. Lipofectamine is the agent used. This technique is less reliable, because you have to hope that the liposome won’t fuse with the lysosomes (where it would be degraded) and that the liposome will make it to the nucleus
HVJ-E transfection
A method of viral envelope transfection. Viral envelopes act like vesicles, but their fusion proteins allow them to fuse with the host cell membranes. The viral envelope is less likely to be endocytosed, it will probably fuse and introduce the foreign DNA. However, the DNA must get to the nucleus. Partial viral particles (non pathogenic) are best to actually get the DNA to the nucleus
Does anything have to be done differently in the lysis of prokaryotic cells?
With prokaryotic cells, you have to consider and disrupt the cell wall
Viral envelopes
Kind of like vesicles, but they also have the presence of fusion proteins in their membrane. This makes fusion with the host cell membrane more likely
Viral particles
Viruses actively infect cells and make their way to the nucleus, making them a good way to deliver foreign objects
