Molecular biology

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In the 1970s the development of techniques that enabled DNA to be cut precisely into specific fragments, and to be joined together enzymatically, paved the road for molecular biology.

DNA characteristics

The strong negative charge on the DNA strands causes electrostatic repulsion that tends to make the two strands repel each other. In the presence of salt, this effect is counteracted by a cloud of counterions surrounding the molecule, neutralising the negative charge on the phosphate groups.

RNA is destroyed under alkaline conditions while DNA is stable.

Removal of 5'-phosphate groups is achieved with an enzyme known as alkaline phosphatase (because of its optimum pH) - one commonly used enzyme is calf intestinal phosphatase (CIP). Removal of the CIP enzyme is best done by phenol extraction and subsequent ethanol precipitation, as heat inactivation is not sufficiently reliable as a method to destroy CIP.

Primer design

A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis. In living organisms, primers are short strands of RNA. A primer must be synthesised by an enzyme called primase, which is a type of RNA polymerase, before DNA replication can occur. The synthesis of a primer is necessary because the enzymes that synthesise DNA, which are called DNA polymerases, can only attach new DNA nucleotides to an existing strand of nucleotides. The primer therefore serves to prime and lay a foundation for DNA synthesis. The primers are removed before DNA replication is complete, and the gaps in the sequence are filled in with DNA by DNA polymerases. In the laboratory, scientists can design and synthesise DNA primers with specific sequences that bind to sequences in a single-stranded DNA molecule. These DNA primers are commonly used to perform the polymerase chain reaction to copy pieces of DNA or for DNA sequencing. https://www.nature.com/scitable/definition/primer-305

Since guanine:cytosine (GC) base pairs have three hydrogen bonds, they are stronger and melt less easily. It is therefore possible to estimate the melting temperature of a DNA fragment if you know the sequence.

Useful enzymes

When supplied with a single deoxynucleotide triphosphate, terminal deoxynucleotide transferase (or terminal transferase) will repetitively add nucleotides to the 3'-OH end of a DNA molecule. This is calling homopolymer tailing.

Linkers and adaptors

Linkers are short synthetic pieces of DNA that contain a restriction site. For example, the sequence CCGGATCCGG contains the BamHI site (GGATCC). Furthermore, it is self-complementary, so you only need to synthesise one strand; two molecules of it will anneal to produce a double-stranded DNA fragment 10 base pairs long. If this is joined to a blunt-ended potential insert fragment by blunt-end ligation, your fragment now will have a BamHI site near each end.

Multiple copies of the linker may be ligated, however, this is not a problem because the subsequent restriction digestion will remove them.

Adaptors are pairs of short oligonucleotides that are designed to anneal together in such a way as to create a short double-stranded DNA fragment with different sticky ends. For example, the sequences 5'-GATCCCCGGG and 5'-AATTCCCGGG will anneal to produce a fragment with a BamHI sticky end at one end and an EcoRI sticky end at the other, without needing to be cut by a restriction enzyme. Ligation of this adaptor to a restriction fragment generated by BamHI digestion will produce a DNA fragment with EcoRI ends that can now be ligated with an EcoRI cut vector.

DNA degradation

Endonucleases attack internal sites in a DNA strand and exonucleases nibble away at the ends.

RNA extraction

http://en.wikipedia.org/wiki/RNA_extraction

Phenol-chloroform extraction http://en.wikipedia.org/wiki/Guanidinium_thiocyanate-phenol-chloroform_extraction

TRIzol http://en.wikipedia.org/wiki/Trizol

Transcriptional activation

http://en.wikipedia.org/wiki/Tetracycline-controlled_transcriptional_activation

http://jaxmice.jax.org/research/tet_intro.html

Studying gene function in eukaryotes by conditional gene inactivation http://www.ncbi.nlm.nih.gov/pubmed/12429690