Molecular Biology of the Cell – PART I
Michel Haring – Lecture 1: Molecular Biology: The Basics (DNA sequencing & PCR)
DNA > RNA > protein > metabolite > phenotype
DNA:
- DNA replication is semi-conservative > every new double stranded molecule consists of one old
and one new strand (left picture)
- DNA synthesis = process of the formation of phosphodiester bonds while hydrolyzing the
matching dNTP molecule (right picture)
- DNA polymerase synthesizes DNA from a double stranded “primer”
- Mistakes in DNA synthesis (normally 1 error in 105 nucleotides) can be restored through:
, • Proof-reading activity of the DNA polymerase complex (1 error per 107 nucleotides left)
• Strand-directed mismatch repair (1 error in 1010 nucleotides left)
• This does not happen in bacteria
- Chemical changes in DNA bases can cause mutation in the DNA
• Depurination = A or G deletion
• Deamination = deletion of amine group: C>T; A>G; G stops replication
• During replication: no (deletion) or any base pair will be incorporated
• Always one strand changed and one strand unchanged
• Changes can be inherited
,Sequencing:
- Dideoxy sequencing (Sanger):
• DNA synthesis while incorporating chain terminators in separate reactions for the bases
A, C, G and T
• The chain terminator (dideoxynucleotide) lacks the 3’ OH necessary for strand
extension
• DNA synthesis will stop when a dideoxynucleotide is incorporated
• Run fragments on a gel and read sequence (manually or automated using a laser and
fluorescence)
- Shotgun sequencing:
• Randomly fragmented genomic DNA (BAC library) > sequencing > assembly by
comparison of overlapping sequences
, • Contigs: assembly of smaller DNA sequences into one continuous strand
• Works well with small genomes that do not have repetitive DNA
- Pyrosequencing:
• Incorporation of a dNTP molecule supplies PPi > converted to ATP > consumed by
luciferase to yield one flash of light
• Also difficult to read repetitive sequences > higher signal when more of the same base
pairs follow up each other
Base order: ATTCCTCGG
- Ion torrent sequencing:
• H+ is released when base pair is built is > measure pH
Michel Haring – Lecture 1: Molecular Biology: The Basics (DNA sequencing & PCR)
DNA > RNA > protein > metabolite > phenotype
DNA:
- DNA replication is semi-conservative > every new double stranded molecule consists of one old
and one new strand (left picture)
- DNA synthesis = process of the formation of phosphodiester bonds while hydrolyzing the
matching dNTP molecule (right picture)
- DNA polymerase synthesizes DNA from a double stranded “primer”
- Mistakes in DNA synthesis (normally 1 error in 105 nucleotides) can be restored through:
, • Proof-reading activity of the DNA polymerase complex (1 error per 107 nucleotides left)
• Strand-directed mismatch repair (1 error in 1010 nucleotides left)
• This does not happen in bacteria
- Chemical changes in DNA bases can cause mutation in the DNA
• Depurination = A or G deletion
• Deamination = deletion of amine group: C>T; A>G; G stops replication
• During replication: no (deletion) or any base pair will be incorporated
• Always one strand changed and one strand unchanged
• Changes can be inherited
,Sequencing:
- Dideoxy sequencing (Sanger):
• DNA synthesis while incorporating chain terminators in separate reactions for the bases
A, C, G and T
• The chain terminator (dideoxynucleotide) lacks the 3’ OH necessary for strand
extension
• DNA synthesis will stop when a dideoxynucleotide is incorporated
• Run fragments on a gel and read sequence (manually or automated using a laser and
fluorescence)
- Shotgun sequencing:
• Randomly fragmented genomic DNA (BAC library) > sequencing > assembly by
comparison of overlapping sequences
, • Contigs: assembly of smaller DNA sequences into one continuous strand
• Works well with small genomes that do not have repetitive DNA
- Pyrosequencing:
• Incorporation of a dNTP molecule supplies PPi > converted to ATP > consumed by
luciferase to yield one flash of light
• Also difficult to read repetitive sequences > higher signal when more of the same base
pairs follow up each other
Base order: ATTCCTCGG
- Ion torrent sequencing:
• H+ is released when base pair is built is > measure pH
