● Molecular Biology and Biotechnology
○ Polymerase Chain Reaction (PCR)
■ Essential Components Required:
● Template DNA, DNA primers (forward and reverse),
deoxyribonucleotides (dNTPs), and a heat-stable DNA
polymerase (e.g., Taq Polymerase)
■ Not required:
● Enzymes like topoisomerase or DNA gyrase are not used in PCR
because high temperatures (94-98 °C) are used to separate the
DNA strands mechanically rather than enzymatically.
○ Recombinant DNA and Cloning
■ Restriction Endonucleases:
● Enzymes used to cut DNA at specific, often palindromic,
recognition sequences.
■ DNA Ligase:
● Convalently seals the phosphodiester backbone between two
distinct DNA fragments.
■ Plasmid Vector Selection (e.g., pUC19):
● Ampicillin Resistance:
○ Growth on ampicillin plates proves successful
transformation (the bacteria took up the plasmid vector
containing the resistance gene). It does not guarantee the
specific gene of interest was cloned into the plasmid.
● Blue White Screening:
○ Successful insertion of a gene disrupts the lacZ gene.
White colonies indicate successful cloning (recombinant
plasmid), while blue colonies indicate an empty vector
(non-recombinant)
○ DNA Sequencing Technologies:
■ Sanger Sequencing vs Next-Generation Sequencing (NGS):
● Both methods utilize DNA polymerase, computers, and
fluorescently labeled nucleotides to read sequences. The
definitive structural difference is that NGS utilizes reversible
terminators, allowing billions of fragments to be sequenced
simultaneously in a massively parallel fashion, whereas
automated Sanger sequencing relies on irreversible
dideoxynucleotides (ddNTPs)
○ Key Biotechnology Enzymes:
■ Reverse Transcriptase:
● Synthesizes complementary DNA (cDNA) using an mRNA
template
■ Cas9:
● An endonuclease that cuts double-stranded DNA at a specific site
matching a single guide RNA (sgRNA) sequence
○ Polymerase Chain Reaction (PCR)
■ Essential Components Required:
● Template DNA, DNA primers (forward and reverse),
deoxyribonucleotides (dNTPs), and a heat-stable DNA
polymerase (e.g., Taq Polymerase)
■ Not required:
● Enzymes like topoisomerase or DNA gyrase are not used in PCR
because high temperatures (94-98 °C) are used to separate the
DNA strands mechanically rather than enzymatically.
○ Recombinant DNA and Cloning
■ Restriction Endonucleases:
● Enzymes used to cut DNA at specific, often palindromic,
recognition sequences.
■ DNA Ligase:
● Convalently seals the phosphodiester backbone between two
distinct DNA fragments.
■ Plasmid Vector Selection (e.g., pUC19):
● Ampicillin Resistance:
○ Growth on ampicillin plates proves successful
transformation (the bacteria took up the plasmid vector
containing the resistance gene). It does not guarantee the
specific gene of interest was cloned into the plasmid.
● Blue White Screening:
○ Successful insertion of a gene disrupts the lacZ gene.
White colonies indicate successful cloning (recombinant
plasmid), while blue colonies indicate an empty vector
(non-recombinant)
○ DNA Sequencing Technologies:
■ Sanger Sequencing vs Next-Generation Sequencing (NGS):
● Both methods utilize DNA polymerase, computers, and
fluorescently labeled nucleotides to read sequences. The
definitive structural difference is that NGS utilizes reversible
terminators, allowing billions of fragments to be sequenced
simultaneously in a massively parallel fashion, whereas
automated Sanger sequencing relies on irreversible
dideoxynucleotides (ddNTPs)
○ Key Biotechnology Enzymes:
■ Reverse Transcriptase:
● Synthesizes complementary DNA (cDNA) using an mRNA
template
■ Cas9:
● An endonuclease that cuts double-stranded DNA at a specific site
matching a single guide RNA (sgRNA) sequence
