DNA Replication and PCR Central Dogma DNA structure

phosphate At physiological p. H phosphate group is charged ? ? What implications does

How are DNA monomers put together Phosphodiester bond what type of bond is this?

What holds helices together ? Hydrogen bonds

What has fallen apart? + DNA = + Boiling water = Denaturation hydrogen bonding

Concepts What do you need to know? • How does DNA polymerase work •

How does DNA polymerase work? • DNA polymerase can only extend preexisting molecules •

Will this system make DNA? d. ATP d. GTP d. CTP d. TTP

Will this system make DNA? yes d. ATP d. GTP d. CTP d. TTP

DNA polymerase can move only in direction: 5’ 3’ d. ATP d. GTP d.

DNA polymerase can move only in direction: 5’ 3’ 3’ 5’ d. ATP d.

Inventor Kary B. Mullis Nobel in Chemistry in 1993 Process for Amplifying Nucleic Acid

Polymerase chain reaction (PCR) a special case of DNA replication in vitro thermocycler

Template DNA polymerase primers unwind DNA d. NTPs, Magnesium, proper p. H and salt

In vitro equivalents • • • Template DNA helicase d. NTPs Mg++ buffer (

Taq polymerase Heat stable Taq: Thermus aquaticus

ALU Insertion Chromosome 16 Alu is an example of a so-called "jumping gene" –

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DNA Replication and PCR

Central Dogma

DNA structure

nucleotides

Pentose (5 Carbon) sugar

phosphate At physiological p. H phosphate group is charged ? ? What implications does this have for DNA molecules?

Bases purines pyrimidines

How are DNA monomers put together Phosphodiester bond what type of bond is this? covalent

What holds helices together ? Hydrogen bonds

What has fallen apart? + DNA = + Boiling water = Denaturation hydrogen bonding is disrupted

Concepts What do you need to know? • How does DNA polymerase work • Concept of molecular machines

How does DNA polymerase work? • DNA polymerase can only extend preexisting molecules • DNA polymerase can move only in direction: 5’ 3’

How does DNA polymerase work?

Will this system make DNA? d. ATP d. GTP d. CTP d. TTP

Will this system make DNA? yes d. ATP d. GTP d. CTP d. TTP

DNA polymerase can move only in direction: 5’ 3’ d. ATP d. GTP d. CTP d. TTP

DNA polymerase can move only in direction: 5’ 3’ 3’ 5’ d. ATP d. GTP d. CTP d. TTP

Molecular Machinery

d. NTPS vs NTPs

Inventor Kary B. Mullis Nobel in Chemistry in 1993 Process for Amplifying Nucleic Acid Sequences Polymerase Chain Reaction Patent Number(s) 4, 683, 202

Polymerase chain reaction (PCR) a special case of DNA replication in vitro thermocycler

Template DNA polymerase primers unwind DNA d. NTPs, Magnesium, proper p. H and salt

In vitro equivalents • • • Template DNA helicase d. NTPs Mg++ buffer ( salt, p. H) • polymerase • • • Template DNA heat (95 o. C) d. NTPs Mg. Cl 2 buffer ( salt, p. H) Taq polymerase

Taq polymerase Heat stable Taq: Thermus aquaticus

Polymerase Chain Reaction

ALU Insertion Chromosome 16 Alu is an example of a so-called "jumping gene" – a transposable DNA sequence that "reproduces" by copying itself and inserting into new chromosome locations. : v First, the inserted Alu is transcribed into messenger RNA by the cellular RNA polymerase. v Then, the m. RNA is converted to a double-stranded DNA molecule by reverse transcriptase. v Finally, the DNA copy of Alu is integrated into a new chromosomal locus at the site of a single- or double-stranded break.