Principles of DNA Isolation/Extraction and

PCR

Ruby Carbonell Paraguison-Alili, PhDMolecular Biologist

College of Veterinary Science and Medicine Central Luzon State University

TJCBTG∞

DNA LOCATION

PRINCIPLES OF DNA ISOLATION & PURIFICATION

DNA/RNA can be isolated from animal

cells, plant cells, bacteria, protozoa or

viruses

Breaking the cells

•Breaking the cell, layer by layer releases the cellular constituents. •Accomplished by lysis of the tissue or any sample and homogenizing in the extraction buffer.

Components ofExtraction/Lysis Buffer

• Tris (Tris-hydroxymethyl aminomethane) buffers the pH of the cells at 8.0.

• EDTA (Ethylene diamine tetraacetic acid) chelates the metal ions,cofactors for DNases; weakens the cell membrane stability.

• Sodium chloride helps in maintaining the osmoticum of the cells. High concentration weakens the membrane integrity.

Components ofExtraction/Lysis Buffer

• Mercaptoethanol cleaves the disulphide bridges of proteins and helps in denaturation proteins.

• Proteinase K – degrade proteins at 56oC

• SDS is a detergent, which also denatures the membrane proteins and disrupts the cell membrane. SDS also helps in inhibition of nucleases.

PCI• The use of Phenol Chloroform-

Isoamyl alcohol) is to remove the proteins, most lipids, and cellular debris that can cause an impure DNA result.

DNA Precipitation• DNA in the nucleoplasm is neutralized by

Potassium acetate. K+ ions bind with negative phosphate backbone of the DNA and shield them. Also, high concentration of NaCl. This favors DNA precipitation from ethanol in cold condition.

• DNA precipitate is settled down as a pellet by centrifugation, purified by 70 % ethanol wash, hydrated in TE buffer.

RNA Isolation/Extraction• Take place in the presence of RNase

inhibitory agents (typically strong denaturants like guanidine salts-guanidine isothiocyanate (GITC), sodium dodecylsulfate (SDS), or phenol-based compounds (e.g.Trizol)

• It is typically prior to and after the isolation when RNA integrity is at highest risk.

DNA and RNA extraction

• You may have the option in choosing the procedure in extracting the DNA or RNA: Using the commercial kit or the conventional method.

Things to remember in RNA Extraction

• This can be problematic when tissues or cells are hard (e.g., bone, roots), when workflows prevent processing immediately after collection (e.g., transport from a site of collection to another location for processing), or when samples are numerous (making rapid processing difficult).

Things to remember• RNA are easily degraded.• RNAses are mostly ubiquitous, there should

be a designated area for RNA work.• RNA area usually uses DNAses and DNA area

uses RNAses..• DEPC – RNAse inhibitor

Questions….

Polymerase Chain Reaction

PCR

• Your definition of PCR…• Developed by Kary Mullis in 1984, leading to

the invention of the Thermocycler/Thermal cycler

• Thermal cycler is complex heatblock wherein different temperatures are set for in vitro amplification or reproduction of target DNA

PCR TargetsThe targets in PCR are the sequences

of DNA on each end of the region of interest, which can be a complete gene or small sequence.

Crucial in primer or DNA marker designing

3 steps in PCR Amplification

• Denaturation• Annealing• Elongation or Extension

DenaturationDenaturation of the DNA is the first

step in PCR, in which the DNA strands are separated by heating to 95°C to 97°C.

Denaturation•Heating separates the double stranded DNA ---Denaturation• Slow cooling anneals the two strands ---Renaturation

Heat Cool

Annealing•Two primers are supplied to bind to the complementary region•As the DNA reaches the appropriate temp, they attach to the two template strands•Optimal temperature varies based on primer length and melting temperature•Typical temperature from 28 to 65 C

Elongation/Extension DNA polymerase duplicates DNA polymerase duplicates

DNADNA Optimal temperature 72COptimal temperature 72C

PCR Requirements and Roles of PCR Reagents

• PCR Mix– Taq polymerase

• Enzyme that extends growing DNA strand of the PCR target 1-2.5 units

– MgCl2

• Provides ions needed for enzyme reaction. Mg is a co-factor of the enzyme .5-2.5mM

– dNTP’s (deoxy-nucleotide-tri phosphate)• Nucleotides (Adenine, Cytosine, Guanine, Thymine)

building blocks for new DNA strands 20-200µM– Buffer - Maintains optimal pH for enzyme (Tris, KCl) pH

8.3-8.8

Roles of PCR Reagents• Primers 0.1-1.0µM

– Anneal to single-stranded DNA template

– Provide initiation site for extension of new DNA

– Forward primer

– Reverse primer

• DNA template– In this case, the product of our DNA extraction

which is not included in the master mix. 1 µg or 10 to 100ng is enough

Considerations• Contamination can easily lead to erroneous results

– Avoid contaminating with DNA or PCR product…• DNA stocks are separated from PCR reagents• Gloves, tips, pipettors, benches• Proper use of instruments, micropipettors

• Carefully measure reagent quantities• Use appropriate cycling conditions• Run with positive and negative controls

Gel electrophoresis is a widely used technique for the analysis of nucleic acids (Agarose gel electrophoresis)

Gel electrophoresis is a procedure that separates molecules on based on size through a gel under the influence of an

electrical field.

HOW ARE PCR PRODUCTS ANALYZED?

buffer

Cathode(negative)

Anode(positive)

wells

DNA

"LAMP" stands for Loop-mediated Isothermal Amplification. This technology was developed by Notomi et al. It is a very sensitive, easy and time efficient method. The LAMP reaction proceeds at a constant temperature using a strand displacement reaction.

Applications1. LAMP is used in rapid diagnosis of viral, bacterial and parasitic diseases.2. It helps in the identification of genus and species-specific parasites.

What is Loop-Mediated Isothermal Amplification (LAMP)?

Developed by Eiken Chemical Co., Ltd. (Japan) Uses 4-6 different primers designed to recognize distinct regions on the target gene. it is expected to amplify the target sequence with high selectivity. Reaction process proceeds at constant temperature (about 60°C- 65°C) May be combined with a Reverse Transcription step to allow the detection of RNA

What is Loop-Mediated Isothermal Amplification (LAMP)?

1. Amplification of DNA takes place at an isothermal condition (63 to 65°C) with greater efficiency.2. Thermal denaturation of double stranded DNA is not required.3. LAMP helps in specific amplification as it designs 4 to 6 primers to recognize distinct regions on the target gene.4. LAMP is cost effective as it does not require sophisticated equipment.5. This technology can be used for the amplification of RNA templates in presence of reverse transcriptase.6. LAMP assay takes less time for amplification and detection.

Advantages

1. Complicated primer design2. Too sensitive and prone to contamination and

small changes in conditions

Disadvantages

1.0

Cut Leaf

Add 50 uL NaOH

Get 10 to 20uL lysate

Add 150 uL Tris-Cl buffer

LAMP Components Purpose Volume: 12.5uL

Nuclease-free Water 5.45

10x LAMP buffer ---Tris-Cl, MgSO4, (NH4)2SO4, KCl

minimize the change in pH making nucleic acid stable 1.25

5M Betaine Betaine was used in the LAMP reaction mixture to reduce base stacking and to increase not only the overall rate of reaction but also target selectivity by significantly reducing amplification of irrelevant sequences

2.5

10uM dNTP mix (deoxy-nucleotide-tri phosphate) Nucleotides (Adenine, Cytosine, Guanine, Thymine) building blocks for new DNA strands 20-200µM

1.0

Primer Mix:F3B3FIPBIPF-loopB-loop

short nucleic acid sequences (generally about 10 base pairs) that serves as a starting point for DNA synthesis

1

8u/uL Bst Polymerase DNA polymerase derived from Bacillus stearothermophillus, able to unwind DNA strands. Its optimum functional temperature is between 60 and 65 °C and it does not require the high temperature (96 °C) step required to denature DNA.

0.25

Reverse Transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription.

0.05

DNA/RNA template Your DNA or RNA extract ~10 to 500ng

After incubation, Sybr Green dye is added to indicate the positive and the negative reactions

LAMP shows a ladder-like pattern of products from the positive control which shows equivalent results with PCR

V 2GP (-) M (-) V 2GP M

ResultsLAMP PCR

The RT-LAMP mechanism• Primers from the target region of the virus genome•Outer primers (yellow and pink) displace the primary strand• Inner primers (dark blue and purple) create loops

• Producing various stem-loop DNA product structures

Questions…