LF aVLMB031 Imaging and Analytical Methods
(Autumn 2023): Methods for nucleic Acid Analysis
Ioanna Papatheodorou
email: 554123@mail.muni.cz
PLAN OF THE LECTURE
• Introduction
• Isolation of nucleic acids
o Isolation with high salt concentrations
o Isolation with phenol – chloroform
• Techniques for nucleic acid analysis
o DNA analysis
o RNA analysis
• “Omics” technologies and nucleic acids
✓ Understand why it is useful to be able to analyze
nucleic acids
✓ How nucleic acid analysis can be used in
research and clinical practice
✓ What the most common methods for nucleic
acid analysis are
✓ How the “omics” technologies contribute to
nucleic acid analysis
INTRODUCTION
The central dogma of molecular biology
transcription
translation
functional role
DNA
RNA
protein
INTRODUCTION
What are the nucleic acids? Let’s revise!
✓ double stranded
✓ more stable → can be preserved for thousands of years in fossils
✓ contains all genetic information and regulatory elements
✓ genes are only a small part of the DNA regions
✓ coding and non-coding regions can offer valuable information
+ hydrogen bonds
+ phosphate
bonds
INTRODUCTION
What are the nucleic acids? Let’s revise!
✓ single stranded
✓ U instead of T
✓ less stable
✓ more “flexible”
✓ can be transported
✓ create secondary structures
✓ can provide information about the coding regions of DNA
✓ can have regulatory roles itself (!) → rRNA, tRNA, miRs
secondary structure with loops
INTRODUCTION
Why is it useful to be able to analyze nucleic acids?
…and what about the clinical practice?
- Biomedical research: investigation of molecular mechanisms (that can lead to novel therapies)
- Translational research: determination of off-target effects of medicine
- Basic research: production of new knowledge → deeper understanding of how the world works
- Forensics: DNA fingerprinting
- Agriculture: species barcoding → detection of adulterated products
- Identification of foreign DNA/RNA (e.g. virus DNA) or mutated genes (e.g. oncogenes, hereditary diseases)
- Paternity tests
- Karyotypes and prenatal testing
- Diagnostics: determination of biomarker levels / risk assessment
Usage of nucleic acid analysis in research and clinic
Genotyping → telling apart wild type (WT – “healthy”) animals from animals
that are genetically modified
Barcoding → determining the origin of products and the presence of foreign /
dangerous elements in them
Determination of the expression levels of biomarkers in patients → prediction
of risk / severity of disease (prognostics / diagnostics)
Determination of the presence / absence of specific gene or SNP → diagnostics
INTRODUCTION
INTRODUCTION
What is the workflow we need to follow in order to study the nucleic acids?
pure DNA
pure RNA
karyotypes
sequencing
qPCR
ISOLATION OF NUCLEIC ACIDS (I)
High salt concentration → makes the proteins and debris precipitate → nucleic acids stay in the supernatant
SDS, EDTA → denature proteins and destroy membranes → help precipitation of proteins / separation of nucleic acids
Isopropanol → organic solvent (nucleic acids can’t be diluted in it) → precipitation of nucleic acids
High salt concentration
rehydration with EtOH
suspension in water
ISOLATION OF NUCLEIC ACIDS (II)
Phenol - Chloroform
Lysis with phenolic reagent
(e.g. TriZOL, ExtraZOL)
fresh
tissue
frozen
cells
sample
centrifugation
QUALITY AND QUANTITY CONTROL
1. Assessment of quantity and purity
✓ Done with Nanodrop or Bioanalyzer instruments
✓ Produces ratios indicative of purity (260/280 & 260/230)
✓ Determines RNA quantity (ng of RNA per μl)
2. Assessment of integrity
✓ Done with electrophoresis
✓ Determines intact of degraded RNA
✓ Intact RNA shows two big bands that correspond
to the two ribosomal subunits
TECHNIQUES FOR NUCLEIC ACID ANALYSIS
SNP / chromosomal analysis – FISH (fluorescent in-situ hybridization)
SNPs
single nucleotide polymorphisms
may correlate with health problems
(e.g. heart disease, metabolic conditions, cancer)
genes
introns
non-coding regions
regulatory elements
karyotype
observation in
fluorescent
microscope
TECHNIQUES FOR NUCLEIC ACID ANALYSIS
Microarrays – use for gene expression
✓ Detection of SNPs
✓ Investigation of both
alleles (in case the
input material is DNA)
✓ Investigation of RNA
transcripts or patient
samples at once
Labeling of samples with fluorescent dyes
control treated
hybridization
to microarray
Lazer hits at
specific wavelength
Calculation of intensity
of each color
TECHNIQUES FOR NUCLEIC ACID ANALYSIS
Southern / Northen blot
isolated DNA isolated RNA
digestion to fragments
(only for DNA)
electrophoresis
(separation of fragments)
transfer
(stabilization of fragments to membrane)
hybridization
(probe binding to specific region)
Visualization
TECHNIQUES FOR NUCLEIC ACID ANALYSIS
qPCR
reverse transcription
from RNA to cDNA
RT-PCR
multiplication of
cDNA of interest
✓ Proper conditions: pH, co-factors
✓ Enzyme
✓ Random primers
✓ dNTPs
✓ Proper conditions: pH, co-factors
✓ Enzyme
✓ Specific primers / probes
✓ Fluorescent agent
TECHNIQUES FOR NUCLEIC ACID ANALYSIS
qPCR
+
✓ Practical, easy to use and optimize
✓ Realtively fast and reproducible results
✓ Extremely sensitive and more specific than serological tests
✓ Wide applicability
From
a clinical perspective:
- Speed depends on laboratory so it may miss the relevant time frame
- Resources available in the clinic for urgent cases or (equipment, trained
staff)
- Diagnosis of infectious disease – false positives/false negatives
From a biomedical research perspective:
-Primers: sequence must be known, primers must be well designed
-Sensitivity/Contamination
“OMICS” TECHNOLOGIES AND NUCLEIC ACIDS (I)
What are the “omics” technologies?
Investigation of the “totality”
Collective characterization of
the DNA, RNA, proteins or
metabolites of samples /
patients
Identification of SNPs
Identification of
alterations in gene
expression
Identification of
biomarkers in the
blood or tissues
Identification of
metabolic
biomarkers
“OMICS” TECHNOLOGIES AND NUCLEIC ACIDS (II)
What is DNA / RNA sequencing?
isolated DNA isolated RNA
Breaking the nucleic acid to fragments
Sequencing of the fragmented pieces
Alignment to the genome
✓ Determination of the nucleotide sequence of the whole genome / transcriptome of a patient
✓ Detection of mutations in the genomic DNA or of alteration in the expression of all genes
✓ Production of the “genomic profile” or “transcriptomic profile” of the patient
…IN CONCLUSION…
✓ Nucleic acids can offer valuable information regarding:
o The expression of various genes
o The presence / absence of polymorphisms connected to diseases
o The origin of products
✓ Research and clinical practice can benefit from nucleic acid analysis via:
✓ Determination of the expression profile of genes
✓ Construction of karyotypes
✓ Hybridization of fragments in microarrays
✓ Some common methods for nucleic acid analysis are
✓ DNA: SNP determination through FISH, Genotyping, Genetic barcoding, qPCR
✓ RNA: RT-PCR, microarrays, Northen blot
✓ The “omics” technologies allow
✓ Scaling-up of the analyses
✓ Production of the information much quicker
✓ Multiple analysis of many DNA/RNA regions
Thank you for your attention!
See you at 27th of October in the lab ☺