Recombinant DNA, Molecular Tools and Bioinformatics

Phylogenetics and sequence search tools

Phylogenetic analysis infers evolutionary relationships from aligned sequences, and the number of candidate tree topologies grows very quickly with the number of taxa. For n taxa, the number of unrooted bifurcating trees is (2n-5)!!, where k!! is the double factorial, the product of all odd integers up to k. The number of rooted trees is (2n-3)!!, because the root can be placed on any of the 2n-3 branches of an unrooted tree. Thus five taxa give 5!! = 15 unrooted trees, and rooted counts are always the unrooted count multiplied by 2n-3.

Statistical support and tree-building criteria are different things. Bootstrap analysis resamples alignment columns with replacement, rebuilds a tree from each pseudo-replicate, and reports the percentage of replicates that reproduce each internal branch, so it tests how reliable a predicted topology is. Tree-building criteria instead choose one tree from many candidates. Maximum parsimony selects the tree that needs the fewest character state changes, so it minimizes rather than maximizes the number of steps, and it uses no probability model. Maximum likelihood and Bayesian methods use probability calculations, and minimum evolution selects the tree with the smallest total branch length.

Pairwise alignment is found by dynamic programming. Global alignment, such as the Needleman-Wunsch algorithm, aligns two sequences over their full length, whereas local alignment, such as Smith-Waterman, finds the best-scoring pair of subregions and resets negative scores to zero. The alignment score is built from match scores, mismatch scores and gap penalties, while the base composition of a single sequence is not part of the optimized score. Secondary structure predictors such as Chou-Fasman and GOR are not alignment methods.

BLAST program names encode the query and database types, and the prefix T marks a translated sequence. BLASTN compares nucleotides with nucleotides, BLASTX translates a nucleotide query and searches proteins, TBLASTN compares a protein query with the six-frame translation of a nucleotide database, and TBLASTX translates both sides in all six frames before comparison. BLAST-family searches target sequence databases, including databases of translated nucleotide sequences, and they do not search ligand or peptide collections.

Remote protein homologs with low identity are detected with PSI-BLAST, which builds a position-specific scoring matrix from the significant hits of one round and repeats the search iteratively.

Each bioinformatic resource has a distinct role. BLAST finds sequences in databanks similar to a query, Bowtie aligns short next-generation sequencing reads to a reference genome, AlphaFold is an AI system that predicts three-dimensional protein structures, and the Protein Data Bank stores experimentally determined structures.

Restriction enzyme recognition sites

Most type II restriction enzymes recognize a short palindromic sequence, which reads the same 5′ to 3′ on both strands, and cut at a defined position within or near it. For a specific k-base site in a random sequence with equal base frequencies, the expected number of occurrences in a molecule of length L is L/4^k. For the six-base palindrome GTCGAC recognized by SalI, a 9000 bp molecule therefore contains about 9000/4096 \approx 2.2 sites, which rounds to 2.

Blunt or sticky ends depend on where the two strands are cut. When both strands are cut at the centre of a palindrome, as in CTG^CAG, CCC^GGG or AG^CT, blunt ends result. When the cuts are staggered, as in G^GATCC for BamHI, single-stranded 5′ overhangs called sticky ends are left, and the BamHI overhang is GATC.

On a circular molecule, two cuts produce two fragments, and the number of distinct bands depends on whether fragments of identical length occur. Fragments of equal size co-migrate in agarose gel as one band. For example, a 4 kb circle cut into two 2 kb pieces, one of which is then halved by a third enzyme, gives 1 kb, 1 kb and 2 kb fragments, but the two 1 kb fragments appear as a single band.

Gene transfer methods

Foreign DNA is introduced into host cells by physical or chemical methods or by a biological vector. Electroporation applies brief electric pulses that open transient pores in the membrane, the gene gun fires DNA-coated microprojectiles into cells, and microinjection delivers DNA through a fine glass needle.

Not every laboratory technique is a delivery method. A DNA microarray is a platform for detecting and measuring nucleic acids with immobilized probes, so it does not insert genes into cells.

Direct DNA transfer introduces naked DNA into cells without a biological vector. Microparticle bombardment, electroporation, and polyethylene glycol (PEG) treatment, which alters membrane permeability for DNA uptake, especially in protoplasts, are direct methods used in plant genetic engineering. By contrast, Agrobacterium-mediated transformation is an indirect method, because the bacterium acts as a vector and transfers T-DNA into the plant genome.

RFLP and molecular markers

Restriction fragment length polymorphism (RFLP) detects inherited sequence differences that create or destroy a restriction site. Genomic DNA from different individuals is digested with the same enzyme, separated by gel electrophoresis, and probed on a Southern blot. A gained or lost site changes the lengths of the fragments that hybridize to the probe, which reveals the variation. Real-time PCR quantifies amplified DNA and reverse transcriptase PCR converts RNA to cDNA, so neither technique reads restriction-site polymorphisms.

Several features allow alleles to be distinguished by RFLP. A difference in the number of recognition sites for a given enzyme changes the number and sizes of the fragments produced. A difference in the number of tandem repeats changes the length of the fragment that carries the repeat array, which is also revealed as an RFLP. Recombination and segregation are meiotic processes that describe how alleles are inherited, and they are not directly visible as fragment differences in a digest.

NMR spectroscopy principles

Nuclei with nonzero spin placed in a magnetic field B_0 absorb radiofrequency energy at the Larmor frequency, \nu = \dfrac{\gamma B_0}{2\pi}, where \gamma is the gyromagnetic ratio. Rearranged, B_0 = \dfrac{2\pi\nu}{\gamma}. For a proton, with \gamma = 2.675\times10^{8} T^{-1} s^{-1}, a spectrometer operating at 600 MHz requires B_0 \approx 14.1 T.

Identifying all functional groups of an unknown compound uses complementary techniques. Infrared spectroscopy detects characteristic vibrations of groups such as C=O, N–H and O–H, and NMR reports the chemical environment of hydrogen and carbon nuclei, so together they identify the groups present. UV-visible spectroscopy detects only chromophores and conjugated systems, and circular dichroism applies only to chiral molecules and reports conformation rather than a functional group inventory.