D1.1

DNA replication 

• Production of identical copies of DNA 

  • New copies will have identical nucleotide sequences to each other and to the original DNA molecule 

  • Carried out in the cell before mitosis and meiosis in eukaryotic cells 

  • DNA replication before mitosis is required for growth and tissue replacement in multicellular organisms 

  • DNA replication before meiosis is required for reproduction 

D1.1.2: Semi-conservative nature of DNA replication and role of complementary base pairing 

DNA Replication is Semi-conservative

• Because new DNA molecules have one parent strand and one newly synthesized strand

• Highly accurate due to complementary base pairing 

D1.1.3: Role of helicase and DNA polymerase in DNA replication 

DNA replication 

• Enzymes are involved in teh process of DNA replication

Helicase

• The enzyme helicase unwinds the DNA double helix 

  • Breaks the hydrogen bonds between complementary nucleotides 

• The two separated strands will act as templates to produce new strands of DNA 

Polymerase

• DNA polymerase moves along eahc strand of DNA linking nucleotides to form a growing chain of nucleotides using the pre-existing strand as a template 

• The complementary base pairs are 

  • adenine– thymine

  • Guanine-cytosine

• The complementary bases are held together by hydrogen bonds 

• DNA replication produces two identical strands of DNA 

• Each strand will have on one new stand 

D1.1.4: Polymerase chain reaction and get electrophoresis as tools for amplifying and separating DNA 

Polymerase Chain Reaction 

• The polymerase chain reaction Annealing: Temperature is reduced to 54°C  which allows DNA primers to bind to both strands of DNA, next to the sequence to be copied.

• The temperature is increased to 72°C which allows Taq DNA polymerase to replicate both strands, starting at the primer. This produces two identical double-stranded DNA molecules.  Both of these are exact copies of the original DNA molecule.

• Steps #1 - #3 are repeated many times to produce many copies of the DNA.

• (PCR) is a method for amplifying (making many copies of) a DNA sequence from a small sample.

• Uses cycles of heating and cooling to amplify a sample of DNA 

• Materials required 

  • DNA to be amplified 

  • Buffer solution 

  • Primer to attach to the DNA to be copied 

  • Taq DNA polymerase - this attaches to the primer and creates a new strand of DNA 

  • DNA nucleotides - these will be linked by taq DNA polymerase to create a new strand of DNA 

• Steps of the polymerase chain reaction include:

  • Denaturation: The DNA sample is heated to 95°C to break hydrogen bonds and separate the two DNA strands.

  • Annealing: Temperature is reduced to 54°C  which allows DNA primers to bind to both strands of DNA, next to the sequence to be copied.

  • The temperature is increased to 72°C which allows Taq DNA polymerase to replicate both strands, starting at the primer. This produces two identical double-stranded DNA molecules.  Both of these are exact copies of the original DNA molecule.

  • Steps #1 - #3 are repeated many times to produce many copies of the DNA.

Taq DNA Polymerase 

• Taq DNA polymerase is obtained from a bacterium (Thermus aquaticus) which is adapted to living in hot springs, so the enzyme is not denatured at the temperatures involved in the PCR process.

Electrophoresis

• Gel electrophoresis is a technique used to separate charged molecules like DNA or proteins.

• Gel electrophoresis can be used to produce DNA profiles, which are commonly referred to as DNA fingerprints.

Gel electrophoresis of DNA 

• Restriction endonuclease enzymes cut DNA into many negatively charged fragments.

• The DNA fragments move from the negative electrode to the positive electrode in an electrophoresis chamber.

DNA profiles 

• A DNA profile is a pattern created from an individual's DNA fragments, using gel electrophoresis.

Electrophoresis and DNA Profiles 

• A sample of DNA is obtained and amplified using PCR

• DNA sample is cut into fragments using restriction endonucleases.

• The DNA is inserted into wells in agar gel which is in a salt solution.

• Electricity is run through the salt solution.

• The (negatively charged) DNA fragments move towards the positive electrode.

• Small (lighter) fragments move faster than bigger (heavier) fragments.

• When a dye is added, a pattern becomes visible. The pattern is the DNA profile.

D1.1.5: Applications of polymerase chain reaction and gel electrophoresis 

Applications of PCR and Gel Electrophoresis 

• Two common uses include:

Forensic investigations: DNA from crime scenes can be processed to produce a DNA profile. The crime scene DNA profile can be compared to DNA profiles from individuals suspected of committing the crime.

• Paternity testing: A child shares half of its DNA with each parent. The bands in the child’s DNA profile will match either the mother or father. It is possible to determine if a man is the child’s father using DNA profiles from the mother, suspected father and child.

D1.1.6: DIrectionality of DNA polymerases

Carbons in deoxyribose 

• Carbons are numbered in deoxyribose 

• Carbon-1 (1’) is the first carbon when moving clockwise from the oxygen atom.

• Carbon-5 (5’) branches off the pentagon shape from carbon-4 (4’).

• Carbon-5 (5’) branches off the pentagon shape from carbon-4 (4’).

• In a nucleotide:

  • The nitrogen base bonds to C-1 (1’) 

  • The phosphate bonds to C-5 (5’).

DNA Replication moves from 5’to 3’

• A DNa molecule has two antiparallel strands of DNA 

• One strand runs from 5’to 3’

• The other strand runs from 3’to 5’

• DNA polymerases can only add to the 3’ end of the growing polynucleotide.

• DNA polymerase moves from 5’ end to the 3’ of a strand of DNA during DNA replication.

D1.1.7: Differences between replication on the leading strand and the lagging strand 

Leading and Lagging Strands

• DNA replication is continuous on the leading strand moving towards the replication fork.

• DNA replication is discontinuous on the lagging strand moving away from the replication fork, producing Okazaki fragments.

D1.1.8: Functions of DNA primase, DNA polymerase I, DNA polymerase III and DNA ligase in replication 

Enzymes involved in DNA Replication 

• Helicase: Unwinds the two strands of DNA by breaking the hydrogen bonds

• Gyrase (topoisomerase): Relieves the strain on DNA strands.

• DNA Primase: Inserts an RNA Primer onto the strands of DNA.

• DNA Polymerase III: Attaches to the RNA primer, and adds DNA nucleotides to a growing polynucleotide in the 5’ to 3’ direction.

• DNA Ligase: Joins Okazaki fragments together.

• DNA polymerase I: Removes the RNA primer, and inserts DNA nucleotides.

DNA Replication 

• Helicase unzips the double helix at the point of origin, by breaking the hydrogen bonds between nucleotides.

• Gyrase relieves the strain during uncoiling.

• DNA primase adds an RNA primer to both strands of DNA.

• DNA polymerase III attaches to the RNA primer, and adds DNA nucleotides in the 5’ to 3’ direction.

• Nucleotides are added using complementary base pairing (Adenine to Thymine, and Guanine to Cytosine).

• Each strand of DNA acts as a template for the synthesis of a new strand.

• DNA polymerase III moves towards the replication fork on the leading strand, and away from the replication fork on the lagging strand.

• Okazaki fragments are formed on the lagging strands as primers must be inserted repeatedly.

• DNA polymerase I replaces the RNA primer with DNA nucleotides.

• Ligase joins the Okazaki fragments together.

• DNA replication is a semi-conservative process, as the daughter DNA molecules have one parent DNA strand and one newly synthesized strand.

D1.1.9: DNA proofreading 

DNA proofreading 

Errors in complementary base pairing are extremely rare during DNA replication. 

However, errors do occur occasionally.

DNA Polymerase III proofreads the growing DNA chain

removes any mismatched bases at the 3’ end, replacing them with the correct nucleotides.