Summary
Summary QDRA
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An English summary of QDRA including principles and additional questions from the practicals.
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1 Basics DNA and RNA1.1 RNA vs DNA
- RNA has Uracil as nucleotide, DNA has Thymine
- DNA lacks the 2’ hydroxyl group (ribose vs deoxyribose)
- RNA is single stranded
- RNA has non-standard nt
- RNA is unstable under alkaline conditions
- In RNA the A-form helix is most abundant
- RNA can leave the nuclear core
1.2 Overview gene expression
1. Transcription: transcribing mRNA of the DNA template strand by the enzyme RNA
polymerase. RNA polymerase binds to the promotor sequence with the help of transcription
factors and unwinds the 2 strands. One of the strands will serve as the template strand
(antisense strand). RNA polymerase reads from 3’ -> 5’ and will generate the mRNA is build
from 5’ -> 3’ attaching nt as it moves along the template strand. Once the elongation is
finished, termination occurs and an mRNA is produced. After mRNA processing from pre-
mRNA (splicing, 5’-cap, 3’-poly-a-tail) it will leave the nucleus.
a. mRNA processing is the reason why DNA and cDNA give different PCR yields (introns are
removed in mRNA)
2. Translation: mRNA finds a ribosome where translation will occur. Each codon will code for a specific
anticodon in a tRNAs which in term will code for an amino acid. The aa are covalently bonded. Folding
and modification of aa chain to produce protein.
a. AUG = Start codon; small ribosomal subunit and initiator tRNA with antistop codon bind to
the mRNA to start = translation initiation complex
b. UAG & UAA & UGA = Termination codons; do not code for aa
1.3 Eukaryotic RNA
- 10-30 pg per cell - 15% low molecular weight RNA
- 80-85% ribosomal RNA (rRNA) o transfer RNA (tRNA), 80 nt
o 18S o small nuclear RNA (snRNA), 20-25
o 5.8S nt
o 28S o microRNA, 21-22 nt
o 5S - 1-5% messenger RNA (mRNA)
Why work with it? To investigate gene expression, determine whether or not gene expression changes when
changing certain parameters, to investigate the biological origin of a biological sample, to investigate the age of
a stain (RNA degradation)
2 DNA and RNA isolation HeLa cells
2.1 HeLa cell line
= an 'immortal' cell line, the oldest and most used human cell line. In comparison to other cell lines,
HeLa cells are the only cells to survive in vitro. They are obtained from cancerous tissues, this makes
them have different genetic trait compared with normal human cells:
- Prolific growth and high growth rate of cells in culture (due to HPV virus in its DNA)
- Disadvantage: Aggressive growth rate can cause contamination, different karyotype from
normal cells
2.2 Tripure: phenol extraction (used for RNA in QDRA)
- Tripure contains monophasic solutions of Phenol and Guanidine Thiocyanate.
1
, - Tripure reagent disrupts cells and denatures nucleases preserving the integrity of RNA and
DNA in the sample. After Chloroform is added to the extract, the entire mix is centrifuged.
The solution contains three phases: a colorless aqueous (upper) phase, a white Interphase
and a red organic phase. RNA is recovered from aqueous phase by isopropanol precipitation.
DNA and protein are isolated from the white interphase and the red organic phase by
ethanol precipitation steps.
- The pH of phenol determines the partitioning of DNA and RNA between the organic phase
and the aqueous phase.
o Neutral or slightly alkaline pH (7-8): phosphate diesters in nucleic acids are negatively
charged, and thus DNA and RNA both partition into the aqueous phase.
o Acidic pH (<7): most proteins and small DNA fragments (<10 kb) fractionate into the
organic phase, large DNA fragments and some proteins remain at the interphase
between the organic and aqueous phases.
- Isopropanol / ethanol precipitation: When the cations and negatively charged nucleic acid
backbone interact, nucleic acids are neutralized, therefore no longer dissolve in water and
precipitate out of solution
o The volume isopropanol is typically half of the volume of ethanol
o Isopropanol is less volatile than ethanol (more difficult to evaporate)
o Salts are less soluble in isopropanol compared to ethanol
Salt concentration and pH determine whether or not RNA or DNA bind: Depending
on the amount of salt the pH changes which can cause the RNA to shift phases. The
lower the salt concentration, the higher the pH.
2.3 Organic extraction (used for DNA in QDRA)
The lipids in the cell and nuclear membranes are removed and the DNA is going to be
purified from cell debris. There are several reagents involved:
- TEN buffer
o Tris-HCl: maintenace of pH stability.
o Chelating EDTA agent: bind divalent cations which will inhibit enzymes such as
nucleases.
o NaCl: neutralises the negative charge on DNA to make it less hydrophilic. (increasing
ionic strength)
- Sodium dodecyl sulfate (SDS): lysis of the cell membrane. = anionic detergent, with its
charge it linearizes proteins in the membrane and removes the protein anions which results
in denaturation. Due to this it aids in the release of binding proteins from the DNA.
Proteinase K: digest contaminating proteins, such as DNAse.
- RNAse A: catalyse the degradation of RNA in the sample.
- Phenol Chloroform Isoamyl alcohol:
o Phenol: denaturation of proteins in the sample and creates the organic phase
(denaturated proteins and lipids) after contribution.
o Chloroform: aqueous phase removes phenol residues and contains nucleic acids.
o Isoamyl alcohol: interphase that prohibits emulsion between the organic phase and
the aqueous phase.
- Sodium acetate: makes sure DNA does not dissolve in water. The compound breaks up into
Na+ and (CH3COO)–. Na+ neutralizes PO3- of the DNA which makes it less hydrophilic.
(increasing ionic strength)
2
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