Introduction to molecular microbiological techniques

Central Dogma

DNA -replication→ DNA -transcription→ RNA -translation→ Proteins

Genetic material

Carries information that code for all necessary components and reactions of life: DNA in bacteria and higher organisms; DNA or RNA in viruses

Replicon

Any DNA molecule that can replicate as a discrete genetic unit: chromosome; plasmids; viral nucleic acids

Gene

A segment of DNA (RNA in some viruses) that encode for a particular protein or RNA (a characteristic)

Gene expression

The process by which information encoded in a gene is converted into a protein or RNA

Genotype

The complete set of genetic determinants of an organism

Phenotype

All the observable characteristics of an organism

Proteins

Built from a repertoire of 20 amino acids (Peptide bonds)

Complex structures in proteins

Secondary, tertiary and quaternary

Function of proteins

Structural, enzyme, signaling, attachment, transport

DNA

Deoxyribose nucleic acid (missing hydroxyl group at 2’)

A polymer of deoxyribose nucleotides are linked together by what bonds?

Phosphodiester bonds

Structure of DNA

Double helix

A-T (2 hydrogen bonds)

G-C (3 hydrogen bonds)

Pyridimies

Cytosine, thymine, uracil

Purines

Adenine, guanine

DNA coding

Three- letter coding system (codons), each codon represents an amino acid (64 codons)

What do codon sequences act like?

Act as signals (promoters, terminators, start codons, stop codons)

Sense/coding strand

Strand carrying genes

Template/antisense strand

DNA strand that is read to build a new, complementary strand

RNA

Ribose nucleic acid

Single stranded U instead of T

A polymer of ribose nucleotides (ATP, UTP, GTP, CTP) are linked by what bonds?

Phosphodiester bonds

Types of RNA

Messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA)

Gene expression (proteins synthesis)

Two steps

• transcription - making a copy of a gene in the form of RNA (mRNA)

• Translation - the process of synthesizing proteins by ribosomes using the code on mRNA

Transcription

Three main steps

• initiation

• Elongation

• Termination

The mRNA has similar sequence to the coding strand but with U instead of T

Initiation (transcription)

RNA polymerase II detect a promoter sequence (gene beginning), DNA unwinds, strands separate, and transcription commences

Elongation (transcription)

RNA polymerase II synthesizes mRNA in 5’ to 3’ by adding nucleotides complementary to template strand

Termination (transcription)

mRNA synthesis ends at the terminator sequence

Translation

Three main steps

• initiation

• Elongation

• Termination

Initiation (translation)

Translation complex forms (ribosome mRNA) start codon (AUG) detected and tRNA brings the corresponding amino acids (methionine)

Elongation (translation)

Codons read one by one and the corresponding amino acids brought by tRNA and added to polypeptide

Termination (translation)

Stop codon detected (UAA, USG or UGA) translation complex dissociates and polypeptide released

Mutation

Permanent change in nucleotide sequence of DNA

Causes of mutation

Spontaneous - (mistakes during replications - 1 in 10^9 bases added)

Chemicals (mutagens)

Radiation (UV, X-rays)

Types of mutation

Point mutations - change in single nucleotides

Segment mutations - deletions, replacements, inversions and insertions

What types of mutations cause a frame shift

Insertion and deletions

Consequences of mutation

No effect (silent mutations)

Altered protein

Gene inactivation (harmful effects - death)

New characteristic

Genetic exchange

Genetic material can be transferred from one bacterium to another by one of 4 methods (Transformation, transduction, conjugation, transposition)

Transformation

Uptake of DNA directly from environment (DNA fragments from lysed cells)

Transduction

Transfer of DNA through a bacteriophage

Conjugation

Transfer of DNA (usually plasmid) through bacterial mating

Transposition

Movement of DNA sequences “jumping” from one DNA molecules to another in the same cell (e.g. chromosome to plasmid)

Recombination

DNA transferred to a bacterial cell by transformation or transduction integrates into chromosomal DNA

Virual replication (??)

Absorption, penetration, uncoats - releasing viral nucleic acids, turns into messenger RNA, make viral proteins from viral RNA, can be assembled to make more of nucleic acids of virus

Class 1 viral

Double stranded RNA, uses host polymerase to make messenger RNA from own DNA

Class II

Has one strand of DNA (+), uses host polymerase

Class III

Double stranded RNA virus, have its own viral polymerase,

Class IV

Positive strand RNA, serve as messenger RNA or reverse transcribe themselves into negative strand RNA

Class V

Class VI of viruses based on replication

Retroviruses, has an RNA and makes DNA out of the RNA

Example: HIV

Nucleic acid extraction

Purpose is to obtain pure DNA/RNA for further lab analysis (e.g. PCR or sequencing)

Contaminates to remove while extracting DNA

Proteins, carbohydrates, lipids, other nucleic acids

Principle/ methods of nucleic acid extraction

Cell lysis, removing protein/carbs, recovering nucleic acids

Cell lysis

Detergents, enzymes e.g. lysozyme (gram + bacteria), sonication, mechanical disruption (bead beating, for gram + bacteria and fungi)

Removing proteins/carbs

Precipitation - chloroform/phenol (pH critical) & salting out

Digestion (proteinase K

Removing nucleic acids

Percipitation (ethanol or isopropanol), solid phase (binding to silica mini-column) (←most common)

Why did we add ethical at end

To recover your dna from aqueous layer

What does pH of chloroform does

Th pH is critical, basic or neutral it will keep DNA and RNA in extract. If pH of chloroform is acidic only RNA would be extracted. Have to use acidic chloroform phenol to just extract RNA

Slide 24

Nucleic acid quantification

Spectrophotometric (e.g. nano drop)

Fluorescence dyes

Spectrophotometric