D1.1- DNA replication

Outline semi-conservative replication

• As one double strand of DNA is replicated, each new double strand of DNA that is produced contains one strand of the original DNA

• This happens because each strand of the original DNA acts as a template for the new strand to build from with accordance to complementary base pairing

Describe the first step in DNA replication in eukaryotic organisms

• Eukaryotic DNA is normally supercoiled by being tightly wound around histones, forming nucleosomes

  • This helps package the DNA and allows it to fit better within the nucleus

• The first step in DNA replication is to unwind the coiled to make the strands accessible to enzymes

• The enzyme helicase then unwinds the double helix and separates the two DNA strands by breaking the hydrogen bonds between the bases

  • This exposes the bases usually protected within the molecule

• Another enzyme called DNA polymerase moves along the separate DNA strand, using them as templates to begin building a new strand of DNA by placing and attaching free nucleotides in a chain

Explain PCR

• Polymerase chain reaction is a technique used to amplify small fragments of DNA

• The desired section of DNA is placed in a reaction chamber that contains

  • Many free nucleotide triphosphates

  • Primers that will allow replication to occur from the desired point

  • Taq polymerase (because it does not denature at high temperatures used in PCR and can therefore continue to function in repeated cycles)

• Denaturation phase: the DNA is heated enough (around 98 degrees Celsius) to break the hydrogen bonds that hold the two strands of the double helix together

• Annealing phase: the sample is allowed to cool to around 60 degrees. The short primer sequence will bond to complementary sequences in the DNA sample

• Extension phase: at around 72 degrees Celsius, the bonding of primers allows Taq polymerase to replicate DNA using the primer as a starting point because DNA polymerases are not able to add the first nucleotide, only extend existing strands

• Once DNA is replicated, the process is repeated.

• Each time the cycle occurs, the amount of DNA doubles, resulting in exponential growth

Describe the process of gel electrophoresis

• Technique used to identify some key features of the DNA through the separation of DNA fragments based on their size and charge

• The technique uses an electrical current to move molecules through a semisolid medium or gel

• Preparation

  • DNA molecules are usually too large for effective separation

  • Restriction enzymes (restriction nucleases) are used to cut DNA at specific sequences

  • Smaller fragments are produced

  • + a gel (jelly-like polymer submerged in a buffer solution) is prepared with wells created within it

• Process

  • DNA fragments are loaded into wells at one end of the gel (closer to the negative electrode)

  • Because DNA has a negative charge, it moves toward the positive electrode

  • Due to the gel being porous, smaller fragments of DNA move quicker while larger fragments move slower

• Interpretation

  • After a set amount of time has passed, a DNA ladder (a mixture of DNA fragments of known lengths is run alongside the samples

  • By comparing the movement of the sample DNA to the ladder, the sizes of the fragments can be determined

Outline how gel electrophoreses can be applied

The fragment patterns produced can be used to create DNA profiles or DNA fingerprints unique to each individual, allowing for identification

Outline the applications of PCR and Gel Electrophoresis

• DNA profiling is the creation of a unique genetic profile by examining variable regions in the genome

• These regions often involve short tandem repeats- short sequences repeated many times in the DNA

  • The number of repeats varies greatly between individuals

• Process:

  • DNA is extracted from cells

  • PCR is used to amplify the target DNA regions (to make enough for analysis)

  • Restriction enzymes cut the amplified DNA at specific sites

  • Fragments of different lengths are generated, depending on the number of repeats

  • Gel electrophoresis separates these DNA fragments by size, producing a band pattern

• Application

  • Forensic investigation

    • Identify whether a suspect’s DNA matches DNA found at a crime scene

  • Paternity testing

    • Comparing the child’s DNA profile to potential parents to determine biological relationships

Explain why the formation of the double helix is antiparallel

• DNA has directionality because of the structure of the nucleotides:

  • Each DNA nucleotide:

    • Has a 5’ end (with the phosphate group)

    • Has a 3’ end (with a hydroxyl group)

  • Each DNA strand

    • nucleotides are connected by phosphodiester bonds between the 5’ phosphate of one nucleotide and the 3’hydroxyl of the next

  • DNA polymerase III can only add a new nucleotide by connecting its 5’ end to the 3’ end of the growing strand

    • Therefore, it builds the new strand in a 5’ to 3’ direction

Describe the formation of the two strands of DNA

• Leading strand:

  • DNA polymerase III follows helicase

  • Replication is continuous (just one primer is placed, and polymerase keeps going

  • goes in a 3’ → 5’ direction

  • Needs only one RNA primer

  • No fragments formed

• Lagging strand:

  • In the direction opposite to the movement of helicase

  • 5’ → 3’ direction

  • Fragments are formed

  • Replication is discontinuous

  • multiple RNA primers are required

Outline the role DNA primase in DNA replication

• Adding RNA primers to the template strands

• Provides a starting point for DNA polymerase III

Outline the role DNA polymerase III in DNA replication

• Main builder of new DNA strands

• Adds free nucleotides according to complimentary base pairing

• Synthesises in a 5 → 3 direction only

• Replicates continuously on the leading strand and discontinuously (through Okazaki fragments) on the lagging strand

• Proofreads the new strand

Outline the role DNA polymerase I in DNA replication

• Removes RNA primers after replication

• replaces them with DNA nucleotides

Outline the role DNA ligase in DNA replication

• seals the gaps between Okazaki fragments by forming phosphodiester bonds

• Joins fragments into one continuous DNA strand

List the enzymes involved in DNA replication

• DNA ligase

• DNA polymerase III

• DNA polymerase I

• DNA ligase