Seismic Method Estimate velocity from seismic data.pptx
Principles of DNA isolation, PCR and LAMP
1. Principles of DNA
Isolation/Extraction and PCR
Ruby Carbonell Paraguison-Alili, PhD
Molecular Biologist
College of Veterinary Science and Medicine
Central Luzon State University
TJCBTG∞
3. PRINCIPLES OF DNA
ISOLATION & PURIFICATION
DNA/RNA can be isolated
from animal cells, plant
cells, bacteria, protozoa or
viruses
4. 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.
5. Components of
Extraction/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.
6. Components of
Extraction/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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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).
12. 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
15. • 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
16. PCR Targets
The 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
17. 3 steps in PCR Amplification
• Denaturation
• Annealing
• Elongation or Extension
18. Denaturation
Denaturation of the DNA is the first
step in PCR, in which the DNA
strands are separated by heating
to 95°C to 97°C.
20. 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
24. 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
25. 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
26.
27. 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
28. 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?
31. "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.
Applications
1. 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)?
32. 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)?
33. 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
34. 1. Complicated primer design
2. Too sensitive and prone to contamination and small
changes in conditions
Disadvantages
36. Cut Leaf
Add 50 uL
NaOH
Get 10 to
20uL lysate
Add 150 uL
Tris-Cl buffer
37. 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:
F3
B3
FIP
BIP
F-loop
B-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
38. After incubation, Sybr Green dye is
added to indicate the positive and the
negative reactions
39.
40. LAMP shows a ladder-like pattern of products from the
positive control which shows equivalent results with
PCR
V 2GP (-) M (-) V 2GP M
Results
LAMP PCR
41. 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
