D1.1 DNA Replication

DNA replication

production of new strands of DNA with identical base sequences to existing strands - suited to be replicated repeatedly due to tis structure

• 2 strands of double helix separate and are both used as templates to guide the polymerization of new strands, adding nucleotides one by one and linking together

• replication fork - site where copying is actually occurring

• semi-conservative - 2 DNA molecules, both with one original strand and 1 newly synthesized strand each

• complementary base pairing - rule that 1 base pair with another, ensures high degree of accuracy in new strands, possible to check assembled base sequence

  • recognize mispairing → cut out and replace incorrect nucleotides, astonishing level of accuracy

biological processes that require DNA replication

• reproduction - offspring need copies of base sequences, parents must replicate DNA to reproduce

• growth and tissue replacement in multicellular organisms - each cell needs full set of organism’s base sequences, DNA must be replicated before cell division (needed for growth and replacing cells)

replisome

functional subunits whose assemblage carries out multi-stage process of DNA replication - proteins helicase and RNA polymerase are essential parts

helicase

unwinding and unzipping DNA

• ring-shaped protein

• separates 2 strands of DNA molecules to act as templates

• moves along DNA molecule, breaking hydrogen bonds between bases, uncoiling helix

DNA polymerase

assembles new strands of DNA using templates from the 5’ to 3’ end

• replisome contains separate DNA polymerase for each strand

• DNA polymerase moves along template strands, adding 1 nucleotide at a time

• brings nucleotides into position where hydrogen bonds could form

• DNA polymerase links nucleotide to end of new strand by making covalent bond between phosphate group of free nucleotide and sugar of nucleotide and the end of the new strand

DNA ligase

binds phosphodiester bonds between nucleotides, goes along the new strand

PCR (polymerase chain reaction)

automated method of DNA replication

• PCR machine (thermal cycler, thermocycler) follow cycle of steps repeatedly, double quantity of DNA with each cycle

• steps in PCR cycler triggered by changes in temperature, only a small quantity of DNA required at the start of the cycle

• DNA amplification - process producing more DNA with specific base sequence

• steps in PCR cycler triggered by changes in temperature

• only selected sequences amplified

• primer - short single strand of DNA designed to bind to DNA at the point where selected base sequence for replication begins, primers added at start of cycling

• Taq (Thermus aquaticus) DNA polymerase - evolved to adapt to high temperatures

typical PCR cycle

1. denaturation

   • breaks hydrogen bonds holding DNA strands together, not covalent bonds in single strands

   • Taq DNA polymerase not denatured

2. annealing

   • cooling allows primers to bind to 3’ ends of target sequence

   • different primers needed for 2 separated strands

   • large excess of primers → rapid binding, prevents single strands of DNA from pairing up again

3. elongation

   • reheated to be optimum working temperature for Taq DNA polymerase, rapid rate of DNA replication

   • new DNA strand assembled by adding nucleotides

gel electrophoresis

separates DNA molecules by length, gel used acts as a molecular sieve, allowing small molecules to pass further through than longer ones as DNA moves away from negatively charged electrode

• short tandem repeats copied, distance traveled by cut up DNA determined by length of DNA molecule

PCR, gel electrophoresis, and coronavirus testing

• process

  • swab taken, virus particles and viral RNA are rinsed in a saline solution to be turned into a liquid sample

  • RNA is converted to DNA using enzyme reverse transcriptase

  • PCR used to amplify specific viral base sequences that are markers of strain of coronavirus being tested for

  • fluorescent markers attached to any DNA produced - fluorescence above target test level means that the test is positive

• advantages

  • sensitive - minuscule quantities can be detected

  • specific - primers can be designed so only 1 strand of the virus is detected

• disadvantages

  • expensive material usually only available in testing laboratories

  • results not available immediately - time taken for thermal cycling

PCR, gel electrophoresis, and paternity testing

• short tandem repeats - base sequences for 2-7 bases that are repeated consecutively, used to distinguish between individuals in DNA profiling

• 13 or more tandem repeats widely used in DNA profiling, unlikely that individuals have same number of repeats of each of these short tandem repeats

• stages to produce a DNA profile

  • obtain DNA sample

  • selected tandem repeats copied by PCR

  • DNA produced by PCR separated according to length of fragment and number of repeats using gel electrophoresis

  • individual’s DNA profile - pattern of bands of DNA procured on gel