A brief but heavily detailed document containing the core subjects in molecular genetics, including key words and their meanings. This will give you a good understanding of molecular genetics at a second year biomedical science level, without having to take in too much information about each indivi...
Denaturation – disruption of bonds and structures in DNA and protein e.g. turning liquid gelatine
into solid jelly. Caused by environment alterations like pH, salt, temp).
Renaturation – Proteins return to normal when normal environment is set
Structure of protein dictates function, and amino acid order determines conformation of
protein.
Monomers are amino acids; proteins are the polymers.
Amino acids contain amino group, carboxyl group, hydrogen atom, variable R group (side chain)
PRIMARY STRUCTURE
R chains differentiate 20 different amino acids – 4 categories are polar (hydrophobic), non-polar
(hydrophilic), acidic, and basic.
Nonpolar (glycine, proline), polar (serine, tyrosine, glutamine),
Acidic (carboxyl acid, negative charge) and basic (hydrogen, positively charged) are electrically
charged
Amino acids exist as stereoisomers (L or D mirror image forms)
mammalian proteins made of L-isomers
Residue or moiety – component amino acid in a polypeptide chain
1⁴ can mean the 4th amino acid etc. etc. with any other number.
Carboxyl group and amine cause condensation reaction
Peptides – links between amino acids.
SECONDARY STRUCTURE
Alpha helix (tight coiled polypeptide backbone core)
Beta pleated sheet formed when two or more polypeptides come side by side
TERTIERY STRUCTURE
3-D conformation. Reveals about function and evolution.
R groups and R groups interact, and R groups and backbones.
Bonds: hydrogen , ionic, covalent (disulphide bridges), hydrophobic (interior of protein)
Proline’s R group connects to amino group and forms natural kink in polypeptide. Tertiary
structure, good for making drugs and vaccines.
X-ray crystallographic studies last years, nuclear magnetic resonance is shorter.
QUATERNARY STRUCTURE
4 tertiary structures coming together (e.g. insulin)
Forms globular and fibrous proteins. not in all proteins.
Globular – water soluble, compact
People with anaemia can struggle with these structures
Fibrous – water insoluble e.g. collagen
Protein binding examples : antibodies, enzymes, neurotransmitters
Protein folding occurs spontaneously and are aided by chaperone proteins (chaperonins),
providing ideal environment for folding.
Conformational change – change in shape once bound to something
Structural layers in the formation of a protein – primary, secondary, tertiary, quaternary
structures. All build up from amino acids in a condensation reaction to form peptide bonds. Each
amino acid could be polar, nonpolar, acidic, or basic in their R groups. Alpha helices and beta
, pleated sheets join in secondary structure. Tertiary 3-D structure are secondary structures joined
by covalent bonds (disulphide bridges), ionic bonds, hydrophobic bonds (interior), and hydrogen
bonds.
10/10/2019
Mobile phase – liquid or gas, a transporter
Stationary phase – impedes different components of the solution to different degrees
Ion exchange chromatography is separation based on charge. Charged molecules are large
proteins, small nucleotides, amino acids. Relies on charge-charge interactions between the
proteins, the two types are: anion exchangers, cation exchangers
Ions – the same charged molecules
Cation exchange chromatography – positively charged molecules are attracted to a
negatively charged solid support. Commonly used resins are: S-resin, sulphonyl group
derivatives,
Anion exchange chromatography – negatively charged molecules are attached to a positively
charged solid support. Common resins are: Q-resin, a quaternary amine.
Oligonucleotide – a big stretch of DNA
Disadvantages of gel flowmetry – proteolysis (the breakdown of proteins) occurs , large size
columns mean large volumes of eluent and higher cost, non-ideal flow around beads
HIS (X6), GST, FLAG TAGs are all specific unique sequences that do not exist in any human
genomes but, when attached to a protein, it allows us to see where the protein is going.
Epitope – short sequence of peptides
Polyclonal antibodies – derived from B cell clones; recognises only a single epitope on an
antigen.
Affinity chromatography has 3 components; matrix, ligands, spacer or covalent link. Has the
highest costs due to the components being so expensive i.e. antibodies cost lots.
17/10/2019
Restriction enzymes – cleaves certain DNA sequences at a particular point.
Dyes to stain DNA will bind to the specific DNA sequences in order to find certain genetic
makeup.
Southern blotting (DNA) – Detection of DNA molecules with transfer. DNA is cut using Res ->
DNA is denatured -> needs to be separated onto an agarose gel or polyacrylamide gel ->
transferred onto a solid support etc. Uses UV light for detection.
Northern blotting (RNA)– Detection of RNA (transcript) molecules for blotting and probing.
RNA extracted from cells fractionated on an agarose gel -> transferred to a nylon (or similar)
membrane -> hybridized to a solution of a radiolabelled cDNA probe corresponding to the
mRNA of interest. Measurement in base pairs.
Western blotting (Proteins) – Detecting proteins following transfer to membranes after
polyacrylamide gel electrophoresis. Proteins extracted through cell lysis, and the proteins
are denatured. Blocking is used to avoid membrane cross-reactions.
KDAs – kilodalton (atomic mass unit)
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