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DNA synthesis & molecular cloning
Concepts
Methods
Applications
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NdeI
BamHI
Enzymatic DNA synthesis: polymerase chain reaction (PCR)
Amplification of up to 20 kbp DNA fragment from pre-existing template
(genomic loci, cDNA library, cloned fragment etc.)
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Jaký je celkový počet polynukleotidových DNA řetězců po n cyklech?
Kolik je nově syntetizovaných polynukleotidových DNA řetězců po n cyklech?
Kolik je nově syntetizovaných cílových polynukleotidových DNA řetězců po n cyklech?
Kolik je cílových dvouvláknových DNA fragmentů po n cyklech?
Kolik se spotřebuje primerů po n cyklech?
Kolik se zabuduje nukleotidů po n cyklech?
Kolik mikrogramů DNA se nasyntetizuje po n cyklech?
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Questions
What is a plasmid?
▪ PLASMIDS are “extrachromosomal” (not part
of the chromosomes), circular pieces of DNA.
▪ Similarly to chromosomes, they are doublestranded,
which means they can easily be
“unzipped” and copied (replicated).
▪ Plasmids use the host’s machinery (DNA
polymerase), but they don’t have to wait for
the host to divide to copy themselves → lots
of copies of themselves.
▪ When the cell does divide, these copies will
get split between the daughter cells, so they’ll
inherit the plasmid as well.
▪ The plasmid can act as a VECTOR – a
vehicle for taking genes we want to deliver
into cells.
Always sequence your plasmid to double-check that the gene is correctly inserted!
Restriction endonucleases (restriction enzymes)
Type I enzymes cleave at sites remote from a recognition site; require both ATP and S-adenosyl-L-methionine
to function; multifunctional protein with both restriction and methylase activities.
Type II enzymes cleave within or at short specific distances from a recognition site; most require magnesium;
single function (restriction) enzymes independent of methylase.
Type III enzymes cleave at sites a short distance from a recognition site; require ATP (but do not hydrolyze it);
S-adenosyl-L-methionine stimulates the reaction but is not required; it exists as part of a complex with a
modification methylase.
Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and glucosyl-hydroxymethylated
DNA.
Structure of DNA
A) A single nucleotide. The phosphate and deoxyribose
sugar form the backbone of DNA. The nitrogenous base
(in this case adenine) is the information-carrying unit
of each nucleic acid.
B) The structure of single-stranded DNA. In nature, enzymes form phosphodiester bonds (blue circles) that
link the 5th position and 3rd position of adjacent deoxyribose sugars. Due to the modular nature of
nucleotides, this chain can grow indefinitely.
Redundancy of the genetic code
• Degeneracy of codons is the redundancy of the genetic code, exhibited as the
multiplicity of three-base pair codon combinations that specify an amino acid
• The genetic code is degenerate mainly at the third codon position
• The genetic code consists of 64 triplet codons specifying 20 canonical amino acids and
3 stop signals
Restriction cloning
The production of exact copies of a particular gene or DNA sequence
using genetic engineering techniques is called gene cloning.
In vitro
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Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz