D1.1 DNA REPLICATION

Define DNA replication, when it occurs, and reasons why

1. DNA replication produces exact copies of DNA molecules

2. Each molecule of DNA produced has identical nitrogenous base sequences

3. It occurs during the s-phase of interphase in the cell cycle

4. It is required for reproduction, growth, and tissue replacement in multicellular organsims

5. Allows for the genetic code to be copied into new cells and passed on to the offspring

Outline the semi-conservative nature of DNA replication and the role of complementary base pairing

1. DNA replication is semi-conservative: each molecule of DNA produced contains one old template and one new strand

2. Complementary base pairing between nitrogenous bases produces:

   • Exact copies of DNA with identical base sequences

   • Adenine pairs/forms hydrogen bond with thymine, guanine pairs with cytosine

   • Allows for genetic information to be replicated

Outline the experiments & hypotheses behind the semi-conservative nature of DNA

1. Watson and Crick proposed the semi-conservative replication, whereas Meselon and Stahl designed the experiment to test the model

2. Bacteria E.Coli was cultured in a heavy isotope of nitrogen (N15), which became incorporated into the DNA’s nitrogenous bases

3. Bacteria were transferred into a medium containing light nitrogen (N14), which was incorporated into then newly made DNA

4. Samples of DNA were taken from each generation

5. Using Density Gradient Centrifugation, the density of the DNA molecules was identified

Recall the enzymes used in DNA replication

1. Helicase

2. DNA primase

3. DNA polymerase III

4. DNA polymerase I

5. DNA ligase

Discuss the role of helicase in DNA replication

1. Breaks the hydrogen bonds between the nitrogenous complementary base pairs between the two strands of DNA

2. Unzips & separates the two DNA strand — each strand becomes a template strand for the new complementary strand to be produced

3. Unwinds the DNA molecule to create a replication fork

Discuss the role of DNA primase enzyme

1. Adds a short RNA primer (a short sequence of RNA nucleotides that are complementary to each template strand)

2. Allows for DNA polymerase to be added in the 5’ to 3’ direction

3. Added to both the leading and lagging strands

4. Doesn’t require an existing strand / 3’ OH group to attach new nucleotides

Discuss the role of DNA polymerase III

1. Adds DNA nucleotides by reading the template strand to add complementary base pairs to the template strand (A/T, G/C)

2. Attaches nucleotides (5’ phosphate group) to the 3’ end of the existing strand

3. Works in the 5’ to 3’ direction

Outline the differences between the replication on the leading strand and the lagging strand

1. The two strands of DNA are antiparallel

2. LEADING STRAND: produced continuously → one RNA primer

3. LAGGING STRAND: are produced in fragments → Okazaki fragments are produced → multiple RNA primers

Discuss the role of DNA polymerase I

1. ReplacesRNAprimase (RNA nucleotides) with DNA nucleotides

2. Exonuclease activity in the 3’ to 5’ direction by removing RNA

3. Works in the 5’ to 3’ to add DNA

Discuss the role of DNA ligase enzyme

1. Seals/nicks the sugarphosphatebackbone

2. Forms covalentphosphodiester bond

Outline DNA proofreading

1. DNA proofreading corrects mistakes in DNA replication

2. DNA polymerases (III and I) have DNA proofreading capabilities & undergo exonuclease activity work in the 3’ to 5’ direction

3. DNA polymerase III removes mismatched nucleotides from the 3’ terminal end and replaces them with a correctly matched nucleotide

4. DNA polymerase I removes the RNA primer/nucleotides and replaces them with DNA nucleotides

Outline polymerase chain reaction

PCR/polymerase chain reaction is used to amplify repetitive sequences (STR/VNTR - short or variable number tandem repeats) of small samples of DNA by:

1. Obtain small sample of DNA (hair, semen, blood, skin)

2. Heat DNA (95 degrees) → breaks hydrogen bonds → separates trands

3. Cools DNA (72 degrees)

4. Adds:

   • primers - bind to the DNA @ lower temps

   • taq DNA polymerase enzyme - heat resistant, doesn’t denature, forms new double-stranded DNA by adding DNA nucleotides by CBP

   • DNA nucleotides( annealing/extending)

5. Repeat heating, cooling, and extending/replicate 30-40 times to obtain million copies of DNA

Outline gel electrophoresis

1. Gel electrophoresis is used to separate proteins or fragments of DNA according to size

2. DNA samples are cut and broken into fragments by restriction endonuclease

3. Smaller fragments travel further

4. A pattern of bands is produced in the gel

5. DNA moves to the positive terminal because it contains negatively charged phosphate groups

Discuss the applications of gel electrophoresis

1. Gel electrophoresis is used in DNA profiling, which is the comparison of DNA → used in forensics, paternity, inheritance, archaeology, or classification

2. FORENSICS: cases of criminal investigations (murder, rape) → obtain reference samples of DNA from suspect → sample obtained from blood, hair, saliva, semen

3. PATERNITY: see who is father/mother → obtain samples from the mother, father, and child

• the bands are analysed for matches between mother and possible father → if some bands are similar, then father (half of the child’s bands will match with mother/father)

Discuss the applications of polymerase chain reaction

1. PCR is used to amplify and produce more copies of DNA

2. Satellite DNA/repetitive sequences/short tandem repeats (STR/VNTR) are used for profiling → statistically impossible to have the same satellite DNA

3. DNA samples are cut and broken into fragments by restriction endonuclease

4. Gel electrophoresis is used to separate the DNA fragments based on their size

5. Smaller fragments travel further, a pattern of bands is produced in the gel → analysed