Module 3: DNA and RNA Extraction

What is the main goal of DNA extraction?

precipitate out proteins while maintaining DNA in solution we will use

What happens during DNA extraction?

DNA extraction procedure (7 steps)

1. Homogenize cells/tissues at 4ºC with sterile equipment

2. Cellular lysis with detergent/lysozyme

3. Add chelating agents ex. EDTA, citrate

4. Add proteinase agents ex. Proteinase K

5. Perform a phenol/chloroform extraction

6. Alcohol precipitation in 70%/100% ethanol

7. Redissolve DNA in Tris-EDTA buffer

*Amount you get depends on amount of starting material

You can study genome with a few cells

DNA extraction: Cellular Lysis

To destroy membranes and release DNA from tissue

DNA extraction: Chelating agents

Chemicals used to block enzymes that will degrade DNA in your sample (DNAses)

DNA extraction: Proteinase Agents

Get rid of whatever you don't want (proteins)

Add enzymes that degrade proteins (particularly Proteinase K)

DNA Extraction: Phenol Extraction

If you put phenol/chloroform, your proteins will separate (precipitate if you centrifuge the sample but DNA will remain soluble)

Easy to take out the protein

To solubilize membranes

To inhibit DNAses

To break down proteins

To precipitate proteins

DNA extraction: Alcohol Precipitation

Add alcohol to DNA (ethanol) in ethanol, DNA precipitates

Forms a white filamentous structure

You can recover DNA pellets

DNA extraction: TE buffer

TE buffer: Provides a good pH and conditions to give DNA optimal conditions

What happens during RNA extraction?

RNA extraction procedure (7 steps)

1. Treat with RNAase inhibitors ex. DEPC

2. Homogenize cells/tissues at 4ºC

3. Cellular lysis with detergent/lysozyme

4. Add RNA solvents, proteinase agents, DNAases

- DNAses can be added to RNA

- If you're analyzing RNA, you can add DNAses to leave RNA aloneterm-28

- Remove proteins the same way as for DNA

5. Perform a phenol/chloroform extraction

6. Alcohol precipitation in 70%/100% ethanol

7. Redissolve RNATreat reage

RNA extraction: Treat reagents with RNase inhibitors e.g DEPC

You will add chemicals that will inhibit RNAses

Very abundant (in skin, in any body secretions)

If you touch dry skin, the RNAses will degrade everything in sample

RNA is prone to degradation

What is the difference between RNA and DNA extraction?

guandinium is added to RNA in order to inhibit RNAses while EDTA is added to DNA. also to add RNAse inhibitors in addition in the first step too since RNA is prone to degradation.

RNA extraction

RNA extraction: Bit more finicky (RNA is more unstable)

RNA is very prone to degradation by RNAses

To break down proteins and inhibit RNAses

DNAse can be added to remove DNA

What allows DNA and RNA and proteins to be purified?

Purification kits using column filtration

column filtration: wash DNA, elute DNA

- More efficient at isolating all three components out of the same sample

- Use of tubes

- Filters like cartridges

- Spin samples through cartridge

- Homogenate goes through dilters, one binds DNA, one binds RNA

- Immobolize DNA or RNA

- Can redissolve what has been immobilized

What is the point of DNA quantification and purity assessment

Wanted to be able to quantify it

How many DNA did we manage to isolate

Are any contaminated?

Contaminated by RNA when we want to get DNA?

What can be used to measure DNA or RNA concentration?

Spectrophotometry

- shine light at a specific wavelength and each cellular component has a distinctive wavelength.

- the amount of wavelength detected = the amount of component.

- out of a single drop (nanodrop)

- to detect the presence of nucelic acids and proteins

DNA samples are considered pure when the A260/A280 ratio is:

Bigger than 1.8

An A260 value of 1.0 represents....

50 µg/mL DNA or

40 µg/mL RNA

If you're purifying DNA, what do you do?

wash and elute from silica membrane

If you're purifying RNA, what do you do?

digest residual DNA and wash/elute RNA

If you're purifying protein, what do you do?

precipitate protein from flow-through, wash pellet, redissolve in Protein Solving Buffer

Absorbance at 260 nm represents

Amount of DNA/RNA

Is DNA or RNA more unstable?

RNA; very fragile

Not only do oyu need to assess amount of RNA isolated

You need to see if it is degraded RNA or if it is intact

How are assays performed to assess RNA quality (is it intact of degraded in fragments)?

Throw samples in a bioanalyzer

Going to electrophoresis your RNA samples

Looks at RNA integrity

Gives out an RNA integrity number

What is an RNA integrity score?

RNA Integrity score (RIN) is assessed by quantifying the abundance of 28S and 18S ribosomal RNA and presence of fragments when RNA is electrophoresed.

10/10 is a perfect score

When a sample is partially degraded (rim score of 5/10)

You can see the peaks of 28S and 18S have broken bands (lower molecular bands)

Shows RNA is partially degraded

What is DNA fragmentation?

DNA is too large to work with after extraction. It must therefore be fragmented into pieces. Restriction enzymes are used to cut the DNA at restriction sites.

Restriction enzymes

Enzyme/endonucleases that cut double-stranded DNA at a specific sequence of nucleotides.

Where do restriction enzymes come from?

bacteria

What are restriction enzymes used for?

1. To generate genomic and cDNA libraries

2. Digestion and separation of genomic DNA for gene analysis

3. Analysis of gene alleles by specifically recognizing single base changes in DNA

4. Insertion of genes in plasmids for gene cloning and recombinant protein production

5. To ligate pieces of DNA together (re-annealing)

T/F: The name of restriction enzymes refers to the organism from which they were first isolated

True

Who discovered restriction enzymes in 1978?

Werner Arber

What is it called when sequences are identical on both DNA strands when read in the 5' to 3' direction?

Palindromic -> Restriction sites are palindromic (what helps enzymes recognize)

How do bacteria protect their own DNA from restriction enzymes?

Methylation of restriction sites

Average size of a restriction site

4-8 nucleotides

Restriction Enzymes: To generate genomic and cDNA libraries

You typically do these when you want to do a genomic project

Sequencing of the human genome.

Library: Your genome broken in pieces, cut in fragments using restriction enzymes and put in a catalogue where you can interrogate whenever you want

Very large genomes, such as human DNA, are usually digested with enzymes that produce long DNA fragments.

That way you have less books to create library that represents your genome

This makes subsequent steps more manageable, since a smaller number of those fragments need to be cloned and subsequently analyzed

Restriction enzymes: To ligate pieces of DNA together (re-annealing)

The DNA products resulting from restriction digestion to form sticky ends may be joined to any other DNA fragments treated with the same restriction enzyme.

libraries to study mRNA

mRNA is UNSTABLE therefore needs to be converted to cDNA first

Use reverse transcriptase

REVERSING MRNA INTO DNA

Restriction enzymes: To ligate DNA pieces

Select restriction enzyme

Cut the end

Treat backbone with the same restriction enzyme

Naturally recombines bc they are complementary bases

Restriction enzymes: Digestion and separation of genomic DNA for gene analysis

DNA fragments are separated according to size using electrophoresis

We create a gel called agarose

Forms a microscopic mesh to separate DNA according to their size

DNA fragments are separated according to size using:

electropheresis

What is the most common gel used for DNA digestion & separation?

agarose

Which gels can also be used to resolve smaller fragments of nucleic acids?

Polyacrylamide gels

Is DNA negatively or positively charged?

negatively

What allows the transfer of DNA to a nitrocellulose of nylon membrane which are more robust, as agarose gels are fragile? (Edwin, 1970s)

Southern blot (Northern blot for RNA)

What is used to visualize DNA (emits fluorescence, highly carcinogenic)

Ethidium bromide

By incubating the gel with a DNA stain (ex. ethidium bromide or biotin). Under UV light, it emits fluorescence.

T/F: Hybridization probes are oligonucleotides of 18-50 bp and labeled with 32P or 35S

True

To identify a specific DNA sequence within DNA fragments, what is the Southern blot membrane incubated in the presence of?

A hybridization probe

High temperature/high salt = high specificity

High stringency

Lower temperature/salt concentration = moderate specificity

Low stringency

What is southern blotting used for?

1. Identifying specific DNA in a DNA sample

2. Isolating desired DNA for construction of recombinant DNA

3. Identifying mutations, deletions, & gene rearrangements

4. Establishing prognosis for cancer/prenatal diagnosis of genetic diseases

5. Diagnosing infectious diseases

6. DNA fingerprinting (paternity, forensics, personal ID)

T/F: Southern blotting is complex, labor intensive, time consuming, and cumbersome

True

What was the first DNA profiling technique that is no longer used?

RFLP analysis

How did RFLP analysis work?

1. DNA is fragmented with restriction enzymes

2. Submitted to Southern blotting

3. Hybridized with DNA probes

In gel electrophoresis, DNA travels from the ______ to the ______ electrode

Negative to positive electrode

Steps of hybridization probe labelling

1. Purify gene probe fragment or synthesize oligonucleotide

2. Alkaline phosphatase treatment of probe to remove 5'-phosphate

3. Polynucleotide kinase transfers phosphate group from donor to 5'-end of probe

4. 5'-end of probe is radiolabelled & gene probe is purified

What do we do if we can't find the gene sequence and therefore can't make a hybridization probe complementary to it? (2 options)

1. Use a related gene, or a gene homolog from a different species

2. Analyze the corresponding protein and sequence the amino acids to reveal the nucleotide sequence

Southern Blot Steps

1. Restriction enzymes cut DNA into fragments

2. Run on gel to separate

3. alkali added to make DNA single stranded

4. DNA Transferred to a nitrocellulose gel filter

5. Filter is exposed to radioactive probe

6.Filter is exposed to photographic film

what does Souther Blot reveal?

1.Identity of the DNA

2. Size of the DNA

3. Abundance of the DNA

RFLP ANalysis

RFLP analysis: first DNA profiling technique. Not used anymore now price and throughput of sequencing have decreased.

Principle: DNA is fragmented with restriction enzymes

Submitted to Southern blotting

Hybrizied with DNA probes

Banding pattern is analyzed for differences between samples.

An RFLP occurs when the length of a detected fragment varies between individuals.

RFLP can be caused by mutations leading to loss of a restriction site. It can also detect insertions, deletions, translocations and inversions.