Jensen Bio D1.1

DNA Replication Overview

Key Terms

• DNA: The molecule that carries genetic instructions.

• Nucleotide: The basic building block of DNA, consisting of a sugar, phosphate group, and nitrogenous base.

• Helicase: An enzyme that unwinds the DNA double helix during replication.

• DNA Polymerase: An enzyme that synthesizes new DNA strands by adding nucleotides.

• Complementary Base Pairs: A-T and G-C pairs that adhere to base-pairing rules during DNA replication.

• Semi-Conservative: Refers to how each new DNA molecule contains one old strand and one new strand.

• PCR: A technique used to amplify small segments of DNA.

• Primers: Short sequences of nucleotides that provide a starting point for DNA synthesis in PCR.

• Taq DNA Polymerase: A heat-stable enzyme derived from the bacterium Thermus aquaticus, used in PCR.

• Denaturation: The first step in PCR where DNA strands separate at high temperatures.

• Annealing: The step in PCR where primers bind to the target DNA sequence at lower temperatures.

• Gel Electrophoresis: A technique used to separate DNA fragments based on size by applying an electric field.

• DNA Profile: The unique pattern of DNA fragments that can be used to identify individuals.

• Restriction Endonuclease: Enzymes that cut DNA at specific sequences, used in cloning and DNA analysis.

• DNA Markers: Specific sequences used as references for analysis and identification.

DNA Replication Process

Required for:

• Reproductive processes in living organisms.

• Growth and development from a single cell to complex multicellular organisms.

• Tissue repair and replacement throughout life.

Process Flow:

1. Unwinding: Helicase enzyme unwinds the DNA helix by breaking the hydrogen bonds between the base pairs, creating replication forks.

2. Synthesis: DNA Polymerase synthesizes new strands by linking nucleotides that are complementary to the template strand. Each new nucleotide is added to the 3' end of the growing strand, ensuring that the process is directional.

3. Result: The outcome of DNA replication is the formation of two identical DNA molecules, each consisting of one original strand and one newly synthesized strand, thus retaining the original genetic information.

Semi-Conservative Replication

• Each resulting DNA molecule is semi-conservative, meaning it contains one original strand and one newly synthesized strand, which is crucial for maintaining genetic fidelity.

• The accuracy of DNA replication is largely ensured by the principles of complementary base pairing, which minimizes errors during the synthesis process.

Techniques: PCR and Gel Electrophoresis

Polymerase Chain Reaction (PCR)

• PCR is a revolutionary technique that allows scientists to amplify specific DNA segments from very small samples, which is especially useful in fields like genetics and forensic science.

• Steps:

  • Denaturation: The DNA strands are separated by heating the reaction mixture to 95°C, breaking the hydrogen bonds between the bases.

  • Annealing: The temperature is lowered to about 54°C to enable the primers to bind to their complementary sequences on the single-stranded template DNA.

  • Extension: Taq DNA Polymerase is then used to replicate the DNA at 72°C, adding nucleotides to build a new strand.

• Taq Polymerase is capable of withstanding high temperatures without denaturing, making it ideal for this thermal cycling method.

Gel Electrophoresis

• Gel electrophoresis is utilized for separating DNA fragments based on their size and charge by applying an electric field to the gel.

• Process:

  • Negatively charged DNA molecules migrate towards the positive electrode, with smaller fragments moving faster than larger ones, effectively separating them.

• This is crucial for creating unique DNA profiles for applications such as forensic investigations, paternity tests, and genetic disorder identification, allowing for detailed comparison and analysis of genetic material.