DESIGNING MOLECULAR PROBES
CELL BIOLOGICAL PROCESSES
− replication:
o templated polymerization of a new strand
o C+G&A+T
o DNA polymerases only extend in 5' to 3' direction
o Okazaki fragments on the 5' strand
− transcription:
o a promoter sequence before
the start site
o RNA polymerase produces
mRNA used later by
ribosomes
o prokaryotes = picture
o eukaryotes = exons and
introns (spliced out at mRNA
maturation), enhancers,
silencers, transcription
factors etc.
− translation:
o ORF:
o open reading frame
o a small subpart of
mRNA that will be translated
o always starts with AUG and stops with UAA, UAG, or UGA
o UTR = untranslated region that doesn't encode for proteins but also consists of exons
o in prokaryotes there are sometimes multiple ribosome-binding sites coding for multiple
proteins → multiple reading frames on one strand
,− protein routing:
o routing of mature proteins to different cellular compartments
o transport through nuclear pores, across membranes, and by vesicles
o NLS (nuclear localization sequence) and NES (nuclear export sequence)
o signal peptide, transmembrane domain, GPI anchors, lipid tails etc.
− RNA flavours:
o mRNAs = coding for proteins
o rRNAs = form ribosome core and catalyse protein synthesis
o microRNAs:
o regulate gene expression
o binding to mRNA to make it double-stranded which is then degraded by the cell =
RNAi (RNA interferences)
o miRNA – endogenous
o siRNA and shRNA – experimental
o tRNAs = adaptors between mRNA and AAs during protein synthesis
o RNA polymerase I = transcribes most of rRNA genes
o RNA polymerase II = transcribes protein-coding genes, miRNA genes, and genes for some
small RNAs
o RNA polymerase III = transcribes tRNA genes, 5S rRNA gene, and genes for many other small
RNAs
− using translation and transcription for research:
MOLECULAR BIOLOGICAL TECHNIQUES
− cloning:
o using nuclear DNA (or a cell) from one organism to create a second organism with the same
nuclear DNA
o cutting a piece of DNA from one organism and inserting it into a 'vector' where it can be
replicated by a host organism → subcloning
− DNA polymerase:
o PCR
o creates DNA molecules by assembling nucleotides
o Taq polymerase = thermostable DNA polymerase isolated from Thermus aquaticus, no
proofreading
, o Pfu polymerase = thermostable DNA polymerase isolated from Pyrococcus furiosus,
proofreading (3’ – 5’ exonuclease activity)
− ligases:
o joining two ends of DNA strands
o if they contain 5’ phosphate groups
o if they ‘fit’ → hard for blunt ends, much more efficient if there is overlap between the ends
− end-modification enzymes:
o Klenow = DNA Polymerase fragment that makes 5’ overhangs blunt by filling in the missing
nucleotides
o Shrimp Alkaline Phosphatase = removes phosphate group at the 5’ end of DNA strands
preventing ligation of these fragments (chemically synthesized oligonucleotides lack 5’
phosphate groups)
o T4 Polynucleotide Kinase = adds (labelled) phosphate group to the 5’ end of DNA strands
enabling detection or ligation
− restriction enzymes:
o molecular scissors that cut double stranded DNA molecules at specific points
o found naturally in a wide variety of prokaryotes
o recognition site = either end of the palindromic sequence (between the two strands)
consisting of around 6 bp
o bacteria use them as defence against bacteriophages → prevent the replication of phage by
cleaving its DNA at specific site, host DNA is protected by methylases adding methyl groups
to A or C within the recognition site
o type 1 = recognize DNA sequence but cut it at random sites as far as 1k bp away from the
recognition site
o type 2 = recognize and cut within the recognition site
o type 3 = enzymes recognize sequences but cut at a different location close to the recognition
site (within 25 bp)
o endonuclease = cuts in the middle of DNA sequence
o exonuclease = cuts only at the end of DNA sequence
o restriction products: 5’ sticky ends, blunt ends (rarely used since ligation is difficult), and 3’
sticky ends
o isoschizomers = restriction enzymes that have the same recognition sequence as well as the
same cleavage site
o neoschizomers = restriction enzymes that have the same recognition sequence but cleave
the DNA at a different site within that sequence
− agents for subcloning:
o not on the exam
o pieces of DNA that can be replicated in the lab
o plasmids:
o circular pieces of double-stranded DNA naturally found in bacteria
o naturally obtained plasmids can carry antibiotic resistance genes, genes for
receptors, toxins, or other proteins
o replicate separately from the genome of the organism
o using pilus encoded by F plasmid to transfer the plasmid to the recipient organism
CELL BIOLOGICAL PROCESSES
− replication:
o templated polymerization of a new strand
o C+G&A+T
o DNA polymerases only extend in 5' to 3' direction
o Okazaki fragments on the 5' strand
− transcription:
o a promoter sequence before
the start site
o RNA polymerase produces
mRNA used later by
ribosomes
o prokaryotes = picture
o eukaryotes = exons and
introns (spliced out at mRNA
maturation), enhancers,
silencers, transcription
factors etc.
− translation:
o ORF:
o open reading frame
o a small subpart of
mRNA that will be translated
o always starts with AUG and stops with UAA, UAG, or UGA
o UTR = untranslated region that doesn't encode for proteins but also consists of exons
o in prokaryotes there are sometimes multiple ribosome-binding sites coding for multiple
proteins → multiple reading frames on one strand
,− protein routing:
o routing of mature proteins to different cellular compartments
o transport through nuclear pores, across membranes, and by vesicles
o NLS (nuclear localization sequence) and NES (nuclear export sequence)
o signal peptide, transmembrane domain, GPI anchors, lipid tails etc.
− RNA flavours:
o mRNAs = coding for proteins
o rRNAs = form ribosome core and catalyse protein synthesis
o microRNAs:
o regulate gene expression
o binding to mRNA to make it double-stranded which is then degraded by the cell =
RNAi (RNA interferences)
o miRNA – endogenous
o siRNA and shRNA – experimental
o tRNAs = adaptors between mRNA and AAs during protein synthesis
o RNA polymerase I = transcribes most of rRNA genes
o RNA polymerase II = transcribes protein-coding genes, miRNA genes, and genes for some
small RNAs
o RNA polymerase III = transcribes tRNA genes, 5S rRNA gene, and genes for many other small
RNAs
− using translation and transcription for research:
MOLECULAR BIOLOGICAL TECHNIQUES
− cloning:
o using nuclear DNA (or a cell) from one organism to create a second organism with the same
nuclear DNA
o cutting a piece of DNA from one organism and inserting it into a 'vector' where it can be
replicated by a host organism → subcloning
− DNA polymerase:
o PCR
o creates DNA molecules by assembling nucleotides
o Taq polymerase = thermostable DNA polymerase isolated from Thermus aquaticus, no
proofreading
, o Pfu polymerase = thermostable DNA polymerase isolated from Pyrococcus furiosus,
proofreading (3’ – 5’ exonuclease activity)
− ligases:
o joining two ends of DNA strands
o if they contain 5’ phosphate groups
o if they ‘fit’ → hard for blunt ends, much more efficient if there is overlap between the ends
− end-modification enzymes:
o Klenow = DNA Polymerase fragment that makes 5’ overhangs blunt by filling in the missing
nucleotides
o Shrimp Alkaline Phosphatase = removes phosphate group at the 5’ end of DNA strands
preventing ligation of these fragments (chemically synthesized oligonucleotides lack 5’
phosphate groups)
o T4 Polynucleotide Kinase = adds (labelled) phosphate group to the 5’ end of DNA strands
enabling detection or ligation
− restriction enzymes:
o molecular scissors that cut double stranded DNA molecules at specific points
o found naturally in a wide variety of prokaryotes
o recognition site = either end of the palindromic sequence (between the two strands)
consisting of around 6 bp
o bacteria use them as defence against bacteriophages → prevent the replication of phage by
cleaving its DNA at specific site, host DNA is protected by methylases adding methyl groups
to A or C within the recognition site
o type 1 = recognize DNA sequence but cut it at random sites as far as 1k bp away from the
recognition site
o type 2 = recognize and cut within the recognition site
o type 3 = enzymes recognize sequences but cut at a different location close to the recognition
site (within 25 bp)
o endonuclease = cuts in the middle of DNA sequence
o exonuclease = cuts only at the end of DNA sequence
o restriction products: 5’ sticky ends, blunt ends (rarely used since ligation is difficult), and 3’
sticky ends
o isoschizomers = restriction enzymes that have the same recognition sequence as well as the
same cleavage site
o neoschizomers = restriction enzymes that have the same recognition sequence but cleave
the DNA at a different site within that sequence
− agents for subcloning:
o not on the exam
o pieces of DNA that can be replicated in the lab
o plasmids:
o circular pieces of double-stranded DNA naturally found in bacteria
o naturally obtained plasmids can carry antibiotic resistance genes, genes for
receptors, toxins, or other proteins
o replicate separately from the genome of the organism
o using pilus encoded by F plasmid to transfer the plasmid to the recipient organism
