Introduction to molecular microbiological techniques (EXAM 2)

Central dogma

DNA -transcription→ RNA -translation→ Proteins

Genetic material

Carries information that code for all necessary components and reactions of life

DNA is in

Bacteria and higher organisms

DNA or RNA is in

Viruses

Replicon

Any DNA molecule that can replicate as a discrete genetic unit

• chromsome; plasmids;viral nucleic acids

Gene

Segment of DNA molecule that in code for a particular protein or RNA

Gene expression

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

Genotype

The complete set of genetics determinants of an organism

Phenotype

All the observable characteristics of an organism

Protein

Built from a repertoire of 20 amino acids

Proteins fold into complex structure

Secondary, tertiary and quaternary

Protein functions

Structural, enyzmes, signaling, attachment, transport

DNA

Deoxyribose nucleic acid, double helix, A-T G-C, polymer of deoxyribose nucleotides linked by phosphodiester bonds

DNA sense/coding strand

The strand that carry genes that are translatable

What are codons and how many?

Codons are the three letter coding system and some sequences act as signals such as promoters, terminators, start codon and stop codon. 64 codons

Antisense/template DNA strand

Used as RNA template, RNA polymerase reads

DNA replication fork

Y-shaped active region where the double helix of DNA unwinds to allow enzymes to create complementary copies of the genetic code

RNA

Ribose nucleic acid, a polymer of ribose nucleotides linked by phosphodiester bonds, single stranded, A-U G-C

Types of RNA

Messenger RNA, ribosomal RNA, transfer RNA

Gene expression (protein synthesis)

Two steps: transcription, translation

Transcription

• initiation

• elongation

• termination

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

Initiation (transcription)

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

Termination (transcription)

MRNA synthesis ends at the terminator sequences

Termination (transcription)

MRNA synthesis ends at the terminator sequences

Translation

• 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 the polypeptide

Termination (translation)

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

Mutations

Permanent change in the nucleotide sequence of DNA

Cause of mutations

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

Chemicals (mutagens)

Radiation (UV, X-rays, etc)

Types of mutation

Point mutations - change in single nucleotides

Segment mutations - deletions, replacements, inversions and insertions

Consequences of Mutations

No effect (silent mutations)

Altered protein

Gene inactivation (harmful effects - death)

New characteristics

Gene 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 the 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 molecule to another in the same cell

Recombination

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

What are the steps of viral replication?

Absorption, penetration, uncoating, transcription/translation, replication, assembly, release

What are the products of viral mRNA?

Structural proteins, enzymes, effects on host cell

What is a class I virus?

Double stranded DNA (dsDNA)

Uses host polymerase → mRNA

What is a class II virus?

Single stranded DNA (ssDNA)

Converted to dsDNA by host polymerase then transcribed into mRNA

What is a class III virus?

Double-stranded RNA (dsRNA)

Requires viral RNA polymerase to make mRNA

What is a class IV virus?

Positive-sense RNA

Genome can function directly as mRNA

Can be translated immediately

What is a class V virus?

Negative-sense RNA

Must use viral polymerase to make =mRNA first

What is a class VI virus?

Retrovirus (+RNA)

Uses reverse transcriptase

RNA → DNA → mRNA

Nucleic acid extraction purpose

Obtain pure DNA/RNA for further lab analysis, (PCR or sequencing)

What are the contaminants that need to be removed?

Proteins, carbohydrates, lipids, other nucleic acids

Methods (cell lysis)

Detergents (SDS), enzymes (lysozymes - gram +), sonication, mechanical disruption (bead beating for gram + and fungi)

Chloroform/phenol method

pH will tell you if you are extracting DNA and RNA together or just RNA

Put RNAase if you dont want RNA in the mix or other enzymes to isolate nucleic acids

Why add ethanol?

To recover DNA from aqueous layer

Chloroform pH basic/neutral

DNA and RNA

Methods (remove proteins/carbs)

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

Digestions (proteinase K - eliminate proteins)

Methods (remove proteins/carbs)

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

Digestions (proteinase K - eliminate proteins)

Methods (recovering nucleic acids)

Selectively binding to what you want

• Precipitation (ethanol or isopropanol)

• Solid phase ( binding to silica mini-column)

Spin column method

What does you use for extraction of RNA

RNAse free reagent and disposables

(Treatment either DEPC) followed by autoclavation

Nucleic acid quantification

Spectrophotometric, fluorescence dyes

Spectrophotometric

Quantity: A260

Quality: A260/A280; A260/A230

Tells us quality and quantity

Fluorecnence dyes

Qubit assays - only quantity

Electrophoresis

Movement of compounds (proteins & nucleic acids) by electricity

Electrophoresis purpose

Separation of proteins or nucleic acids in a mix based on: molecular weight, charge, shape/size; negative → positive

Slab electrophoresis

Agarose gel electrophoresis: nucleic acids

SDS PAGE: proteins

Capillary electrophoresis

Employed in DNA sequencing

DNA cloning

In vivo, cut DNA in specific sites: prepare fragments for insertion site; the more bacteria replicates the more the gene of interest is replicated too