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Summary DNA technology (NWI-MOL027)
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Summary for the course DNA technology.
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DNA technology summaryDNA
A-T, G-C pairing
AT 2 hydrogen bonds, GC 3 hydrogen bonds (more stable)
Normal, relaxed B-form:
Right handed double helix (when not denatured)
10.5 bp per helical winding
3.4 Å per base pair
36 Å per helical winding
Stability of DNA is determined by:
Hydrogen bonding
Base stacking interactions
Charge of phosphate backbone
Tm: temperature at which 50% primer DNA is double stranded
Rule of thumb for Tm = 2 x (A+T) + 4 x (G+C)
Influences on Tm:
- Formamide: interferes with base stacking interactions
- Cation concentration: higher cation concentration neutralizes negative charge on phosphate
backbone therefore increases stability
- pH: a high pH (>10) deprotonates the DNA bases no H-bonding possible
RNA
In contrast to DNA, it has a 2’OH which makes it way less stable than DNA and it can form hairpin
structures as it is most of the time single stranded.
RNA is synthesized during transcription from the ‘leading’ or ‘template’ strand.
mRNA: messenger RNA
tRNA: transfer RNA
rRNA: ribosomal RNA
mtRNA: mitochondrial RNA
miRNA: short RNA fragments that form hairpin loops and interfere with replication, transcription and
translation of ssDNA (not included to this course)
,central dogma
DNA replication
DNA synthesis: 5’3’
Reading of leading strand: 3’5’
Lagging strand: 3’5’ reading but oriented 5’3’ so synthesis of Okazaki fragments
DNA synthesis done by DNA polymerase (DNA pol I, II or III) in organisms
DNA polymerase needs 3 things:
- template strand
- primer with free 3’OH
- dNTPs
positive tension ahead of DNA polymerase, negative tension behind DNA polymerase
DNA synthesis by DNA polymerase:
Sometimes mispairing of bases (e.g. C-A and T-G) can be caused by sporadic imino form of A or C
or sporadic enol form of T or G.
Therefore, all DNA polymerase exhibit 3’5’ exonuclease activity (proofreading):
,Enzymes involved in DNA replication:
Helicase: unwinds DNA
Gyrase: is a topoisomerase II that relaxes positive supercoils release of tension ahead of
the DNA polymerase so that replication can continue
Primase: adds DNA primers to lagging strand (used by pol III) to synthesize dsDNA
RNA primers removed by nick translation
Ligase: ligates remaining nicks after nick translation
, Nick translation is a form of 5’3’ exonuclease activity to remove RNA primers incorporated in the
DNA that is transcribed from the lagging strand. Only DNA polymerase I can do this
PCR: polymerase chain reaction
Allows controlled amplification of (specific) fragments of DNA (by the choice of the primer)
This is used to:
- quantify the amount of DNA that was present in the sample (quantitative PCR)
- amplify the obtained DNA for agarose gel electrophoresis (cloning result, forensic
genotyping)
- addition of restriction sites by addition of a specific sequence at the 5’ site of the primers
(cloning)
amount of DNA obtained after n cycles with a starting amount of x DNA fragments = x 2 n
Taq polymerase is used for PCR, as this is a polymerase originating from bacteria in hot water springs,
so its activity is not decreased by denaturation at high temperatures (useful as high temperatures are
required for PCR)
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