Orderly and clear summary of lecture 3 Molecular cloning and genome sequencing from the book "Medical Genomics in biomedical sciences by Sander Groffen". All the summaries of the lectures match with this book therefore you will easily pass your exam. This will save you a lot of time. I passed this...
Medical genomics – Lecture 4 – Timeline of genome sequencing
The dawn of genetics – from heritability to DNA structure
• Darwin publishes ‘on the origin of the species’
• Mendel’s experiment on plant genetics
• Friedrich Miescher isolated infamous substance (DNA) from leukocytes.
• Albrecht Kossel named the builing blocks of the DNA (C,G,A,T)
• Thomas Hunt Morgan discovered that genes are located in chromatins. The
genetic inheritance of fruit fly D. Melanogaster is linked to the sex of individual
flies.
• Erwin Chargaff conclude that CG contect differs from species, but AT remains
the same
• Watson-Crick modal for double helix
• Semi-conservative DNA replication model; each parental DNA strand remains
attached to a newly synthesized daughter strand upon mitosis
Sequencing (determine the nucleic acid sequence) – The first generation
• First sequence obtained (of alanine-tRNA)
2 methods for base-by-base sequencing;
1. Sanger method; use dideoxy nucleotides --> are
modified nucleotides they have no O group to 3’. So
terminate the chain.
Do this with different nucleotides and sequence can be read by gel
electrophorese
The band will correspond to the chain length.
You want genome of 100 base pairs. Use 4 Eppendorf vials for each base pair
(GATC), gel has 4 lanes one for each band. Read from bottom to the top
2. Cycle sequencing; improved the yield of DNA products generated from a given
amount of template DNA in a single reaction. NOT comparable to PCR
because only 1 primer is used. So, product is linear. (20 cylci = 20 copies)
PCR used 2 primers; forward and reverse. So, product is
exponential. (20 cycli = 2^20 copies)
For drawing see the paper (schrift)
2 techniques for base to base replication;
1. Four color/one lane sequencing technique; the dideoxy
nucleotides are labeled with fluorescent dye. Instead of
having to perform 4 sequence reactions, all four
nucleotides could now be read from a single sequence
reaction
, 2. Electrophorese; method makes it possible to have
multiple samples scanned by a detector. Not needed
to prepare large sequencing gels.
Molecular cloning in first-generation sequencing
Molecular cloning techniques; to generate many identical
copies. In Sanger sequence many identical copies are
needed.
1. DNA fragment is implanted into a cloning vector
(plasmid)
2. Plasmid inserted in bacterial cell by heat schok
3. Cell is growing to form colonies
4. Colonies are harvested (geoogst) and DNA is isolated
From colonies to polonies
Other technique to generate many identical copies; PCR (=polymerase chain
reaction)
Before PCR amplification (vermeerderen) could be applied to sequencing a few
obstacles had to be overcome;
1. PCR uses primers, the sequence of which has to match the template DNA.
How can you amplify a sequence that yet has to be sequenced?
2. A normal PCR used a liquid where all produced DNA copies will mix. This
implies that you can only amplify a single unique template DNA per reaction
For the first; primers is developed
For the second; methods developed that separate the unique templates, so a PCR
could use more templates.
Polonies; combination of colonies and polymerase.
This is the foundation (fundering) of NGS (next generation sequencing)
Next generation sequencing (NGS)/Massively parallel sequencing (MPS)
Difference between NGS and FGS it that samples can be analyzed simultaneously in
a single reaction mixture.
The reaction take place in a collective reaction volume while the process of each
reaction is monitored separately --> large amount of data is analyzed --> companies
invest into technology for NGS
Typical scheme;
• Sample preparation (=library construction)
DNA is isolated from cells of interest. In the case of genomic DNA, it is fragmentated.
--> The DNA fragments are edited by ligation adapter (small piece of double-stranded
DNA with a precisely known sequence) make adapter ligation successful;
a. DNA polishing; DNA fragments are treated to remove single-stranded
overhangs. All fragments now have blunt end (gelijk)
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