GENETICS LAB LE 1

• Central dogma of molecular genetics

  Directional flow of genetic information from DNA to RNA to protein
• Genetic code


  • Written in linear form, uses ribonucleotide bases as letters
  • Sequence derived from complementary nucleotide bases in DNA
• Codon


  Each group of 3 ribonucleotides, specifies 1 amino acid, making the code nearly unambiguous
• Genetic code


  • Degenerate (an amino acid can be specified by more than 1 codon)
  • Contains 1 start and 3 stop signals that initiate and terminate translations, respectively
  • Has no internal punctuations
  • Nonoverlapping
  • Colinear; the order of codons in the mRNA determines the order of amino acids in the encoded protein
  • Nearly universal
• Transcription


  1. RNA molecules are synthesized on a DNA template

  2. Results in an mRNA molecule complementary to the gene sequence of one of the two strands of the double helix

  3. Each triplet codon in the mRNA is, in turn, complementary to the anticodon region of its corresponding tRNA which inserts correct amino acid into the polypeptide during translation
• RNA acts as intermediate in the flow of information from DNA to proteins
• DNA replication occurs in the nucleus, transcription occurs in the nucleus, translation occurs in the cytoplasm or ribosomes
• RNA polymerase


  • Has the same general substrate requirement as does DNA polymerase (but has ribose as substrate nucleotide instead of deoxyribose form of sugar)
  • No primer required to initiate synthesis
• Template strand


  Template for RNA polymerase
• Coding strand


  Complementary DNA strand; has the same 5' to 3' nucleotide sequence but with U substituted for T in the RNA
• Consensus sequences


  • DNA sequences that are similar (homologous) in different genes of the same organism or in one or more genes of related organisms
  • TATAAT/Pribnow box - found in promoter region
  • TTGACA - promotor region: initiation site where RNA polymerase will bind
  • Mutations in either region diminish transcription, often severely
• Transcription


  1. Initiation: Once RNA polymerase has recognized and bound to the promoter, DNA is locally converted from double stranded to open structure to expose its template strand

  2. Elongation: Subsequent ribonucleotide complements are inserted and linked together by phosphodiester bonds as RNA polymerization proceeds (in a 5' to 3' direction in terms of the nascent RNA)

  3. Proofreading: Performed as it adds each nucleotide, in case a complementary mismatch is recognized, the enzymes back up and remove the mismatch, then reverses direction and continues with elongation

  4. Termination: Intrinsic termination (hairpin secondary structure): 80% of E. coli genes, Rho-dependent termination (⍴ termination factor): 20% of E. coli genes
• Transcription in eukaryotes occur in the nucleus
• RNA polymerase in eukaryotes


  • RNA polymerase I and III: transcribe transfer RNA (tRNA) and ribosomal RNA (rRNA)
  • RNA polymerase II: transcribes protein-coding genes (mRNA)
  • 4 different types of cis-acting DNA elements regulate the initiation transcription by RNA polymerase II: core promoter, proximal-promoter element, enhancer, silencers
• In bacteria, transcribed DNA to mRNA is immediately translated into amino acid sequence, but eukaryotic mRNAs require significant alterations before they are transported to the cytoplasm for translation
• Post transcriptional modifications in eukaryotes

  1. Caps and tails: 7-methylguanosine (m^7G) cap is added at the 5', poly-A polymerase catalyzes the addition of a poly-A tail at the free 3'-OH group at the end of the transcript

  2. RNA Splicing: Introns (intrageneic region) are removed, Exons (expressed region) are retained to produce mature mRNA
• Introns


  Non-coding internal sequences present in pre-mRNAs but are removed to produce mature mRNA
• Why introns exist

  • Alternative splicing: production of different mature mRNAs from the same pre-mRNA
  • Exon shuffling: introns have been an important part of the evolution of genes
  • microRNA (miRNA): introns can contain non-coding RNA that regulates gene expression
  • Regulates transcription: intronic sequences in the DNA frequently harbor cis regulatory elements such as enhancers and silencers
• Transcription (DNA to mRNA)


  1. Instructions to make proteins are contained in DNA

  2. DNA contains genes, which contain a region that codes for an RNA molecule, a promoter, and a terminator

  3. Promoter region functions as a recognition site for RNA polymerase to bind

  4. Binding causes the DNA double helix to unwind and open

  5. During elongation, the RNA polymerase slides along the template DNA strand, linking nucleotides to the 3' end of the growing RNA molecule

  6. Once the RNA polymerase reaches the terminator, the messenger RNA transcript is complete and the components dissociate

  7. The messenger RNA includes coding exons and non-coding introns

  8. Intron splicing by the spliceosome removes the introns and joins the exons to produce a mature messenger RNA strand
• Genetic code

  Nitrogenous bases are grouped in three letter codes called codons, most codons code for specific amino acids, there are 4 special codons: one that codes for start, three that code for stop
• Agarose gel electrophoresis


  • Quantitative method to determine if you have nucleotide products and can also be used to determine the sizes of DNA fragments
  • Solid bands represent DNA fragments of uniform lengths, faster moving bands represent shorter DNA fragments
• Sheering of DNA


  Cutting up DNA into smaller fragments of variable lengths creating a paint brush stroke on the gel
• DNA quantification


  Determine average concentrations of extracts or mixtures, helps determine purity
• DNA quantification methods


  1. Spectrophotometry: takes advantage of the natural absorbance of nucleotides of light with a wavelength of 260 nm, concentration is derived through Beer-Lambert Law

  2. UV fluorescence: nucleotides can be made to fluoresce by exposing them to fluorescent substances that intercalate with nucleotides
• Spectrophotometer


  Instrument that measures the amount of light that passes through a medium at a specific wavelength, according to Beer's law the amount of light absorbed is directly proportional to the concentration of the absorbing material
• DNA quantity and quality from spectrophotometer


  • DNA excites at 260˚A, 1 OD260: 50 ug/mL DNA
  • Proteins excite at 280˚A, 260:280 ratio used to evaluate purity (ideal 1.8-2.0)
  • Solvents, EDTA, phenol, alcohol and carbohydrate contaminants excite at 230˚A, 260:230 ratio also a measure of quality (expected 2.0-2.2)
• DNA ladder


  Used as a reference to estimate the size of unknown DNA molecules in gel electrophoresis
• Nucleotides optimally absorb light at a wavelength of 260 nm, so to determine the concentration of a nucleotide solution, it must be run at a spectrophotometer under these conditions
• DNA ladder


  Used as a reference to estimate the size of unknown DNA molecules
• DNA separation in gel electrophoresis


  1. DNA separated based on their mobility in an electrical field through the gel (cathode and anode)

  2. DNA ladder applied as a reference
• DNA ladders


  Widely used in gel electrophoresis as a reference point or molecular-weight size marker to determine the approximate size of unknown DNA fragments
• DNA ladder selection

  Users choose their DNA ladders based on the range of DNA sizes. A 1 kb recommended for determining the size of double-stranded DNA
• If an equal amount of distilled water in a ratio of 1:1, only 1/2 of the solution is DNA and the 1/2 was water the yield should be 2x. if only 1 part DNA: 9 parts water, there is only 1/10 of the total solutions as DNA, the yield should be 10x
• Translation


  The last process in central dogma
• Central dogma processes

  • Replication = DNA
  • Transcription = mRNA
  • Translation = protein
• mRNA


  Holds the codon
• rRNA


  Ribosomal RNA
• tRNA


  Holds the anticodon
• Translation


  Biological polymerization of amino acids into polypeptide chains which occurs in ribosomes (workbenches for polypeptide synthesis)
• Transfer RNA (tRNA)


  Specific molecules that adapts present genetic information as triplet codons in mRNA to their corresponding amino acids