dna replication
D1.1 DNA replication
Continuity and change—Molecules
Standard level and higher level: 2 hours
Additional higher level: 2 hours
Guiding questions
• How is new DNA produced?
• How has knowledge of DNA replication enabled applications in biotechnology?
Recommended prior learning: A1.2 Nucleic acids
SL and HL
D1.1.1—DNA replication as production of exact copies of DNA with identical base sequences
Students should appreciate that DNA replication is required for reproduction and for growth and tissue
replacement in multicellular organisms.
DNA replication is the process of producing exact copies of DNA with identical base sequences. This is
required for reproduction in order to pass genes to offspring, in addition to growth and tissue repair in
multicellular organisms. Although the complexity of the process differs between prokaryotes and
eukaryotes, the general mechanism is similar.
D1.1.2—Semi-conservative nature of DNA replication and role of complementary base pairing
Students should understand how these processes allow a high degree of accuracy in copying base
sequences.
Semi-conservative replication entails the separation of the two parental strands and using each one as a
template for the synthesis of the new complementary strand. This results in two DNA molecules, each with
one original strand and one new strand (the parent DNA molecule is thus only semi/half conserved). The
reason why many organisms have evolved such a mechanism of replication remains unclear.
In order to reduce the number of errors during replication, the chemical structure of the 4 DNA nucleotides
allows bonds to occur only between pairs of A + T and C + G (complementary base pairing). So, if an A
were to be added to a C molecule during replication, it would be rejected as they are not chemically
compatible/complementary, thus errors are reduced, and a high degree of accuracy is achieved.
D1.1.3—Role of helicase and DNA polymerase in DNA replication
Limit to the role of helicase in unwinding and breaking hydrogen bonds between DNA strands and the
general role of DNA polymerase.
DNA replication is heavily dependent on enzymes, for instance:
• The enzyme helicase (donut-shaped) unwinds (separates) the two DNA strands at origins of
replication (regions of DNA where replication is initiated) by breaking the hydrogen bonds between
complementary base pairs
• The enzyme DNA polymerase adds nucleotides one-by-one to the growing template strand
through complementary base pairing by utilizing energy from ATPD1.1.4—Polymerase chain reaction and gel electrophoresis as tools for amplifying and separating DNA
Students should understand the use of primers, temperature changes and Taq polymerase in the
polymerase chain reaction (PCR) and the basis of separation of DNA fragments in gel electrophoresis.
When collecting DNA samples, often the number of molecules is too small for analysis, so it needs to be
amplified. This is done through the Polymerase chain reaction (PCR), which involves the following steps:
1. 2. 3. Denaturation: in order to separate the two strands without enzymes (to reduce costs), the DNA
sample is exposed to high temperatures in order to unwind the strands
Annealing: the sample is cooled in order to anneal (add) the RNA primers which are needed for
initiating DNA polymerase activity
Synthesis: the sample is warmed again and Taq polymerase (a special type of DNA polymerase
derived from the bacterium Thermus aquaticus which can withstand high temperatures, thus
function faster than its human counterpart) is added along with nucleotides in order to carry out
DNA replication. Each cycle (replication) exponentially increases the amount of DNA, so around 10-
13 cycles are done per sample to ensure sufficient quantity
Gel electrophoresis is a biotechnological tool used to separate DNA fragments (or other biomolecules like
proteins and RNA) based on size and charge. However, since all DNA molecules have the same charge per
mass, electrophoresis separates DNA fragments by size only. This is useful in order to isolate
chromosomes and specific genes from the entire genome for analysis. Electrophoresis is usually done after
a PCR in order to collect enough quantities DNA.
The apparatus for gel electrophoresis involves a tray (box) containing a gel with a cathode (negative
terminal/pole/electrode) on one side and an anode (positive terminal) on another side. The DNA samples
are collected using a pipette and inserted in slots (wells) on the cathode (negative) side with a fluorescent
marker to better visualize them. Once power is supplied to the electrodes on both sides, an electrical field is
applied to the DNA molecules (which are negatively charged) and causes them to move towards the anode
(positive terminal). Shorter fragments move further due to their smaller size. A well-defined and clear
line/strip of DNA on the gel appears after a while and is called a band. Each band contains a group of
same-sized DNA fragments as individual ones are too small to be seen (hence why a PCR is needed
beforehand).
The type of gel and strength of electrical field influences the distance the DNA fragments travel. Usually,
one well is reserved for a DNA ladder, which is a standard reference that contains known lengths of DNA
fragments in order to compare the sample to it. Figure 1(B) does not specify which direction the DNA
fragments are travelling to, but the ‘hooks’ towards the end of each band point upwards, indicating that the
wells are up and so the direction of DNA travel is downwards towards the anode.
Common units of measuring DNA fragment length are bp (base pair) and kbp/kb (kilo base pairs).
A
A
B
Figure 1: (a) gel electrophoresis (Rogers), (b) DNA ladder (Tan Laboratory, Penn State University).D1.1.5—Applications of polymerase chain reaction and gel electrophoresis
Students should appreciate the broad range of applications, including DNA profiling for paternity and
forensic investigations.
PCR tests are commonly used to detect viruses (like COVID-19) and other diseases. They are also useful
when conducted before gel electrophoresis, like paternity tests in which the biological parents of a child are
identified, and in DNA profiling for forensic investigations, both of which depend on STRs.
Short Tandem Repeats (STRs) (also known as microsatellites) are DNA base sequences between 1-6
base pairs in length that are repeated consecutively and form series spanning a maximum of 100
nucleotides. They are found widely in both prokaryotes and eukaryotes, and are scattered almost evenly
across the human genome to make up around 3% of the entire genome. Residing in mostly noncoding
regions, STRs have a high mutation rate and thus are highly variable between individuals, so it is very
unlikely that two people have the same STR lengths.
For paternity tests, some STRs are passed to the child from both parents. Multiple STR loci from the
parents and child undergo PCR and then are separated by electrophoresis before being compared. The
person with the greatest number of similar bands to the child is the biological parent. For DNA profiling
during forensic investigations, the suspect with the greatest number of similar bands to the DNA sample
collected at the crime scene is the criminal.
NOS: Reliability is enhanced by increasing the number of measurements in an experiment or test.
In DNA profiling, increasing the number of markers used reduces the probability of a false match.
Increasing the number of STR loci used for DNA profiling or paternity tests reduces the probability of a false
match, as the likelihood of having all chosen STR loci be similar will be reduced.
Additional higher level
D1.1.6—Directionality of DNA polymerases
Students should understand the difference between the 5' and 3' terminals of strands of
nucleotides and that DNA polymerases add the 5' of a DNA nucleotide to the 3' end of a strand of
nucleotides.
DNA polymerases are a family of enzymes involved in DNA replication. DNA polymerase III is the main
enzyme that adds nucleotides to synthesize new DNA, and it can only do this in a 5’ to 3’ direction by
connecting the 5’ OH group of a free/new nucleotide with the 3’ OH pentose group of a nucleotide on the
parental strand. This is evolutionarily advantageous because the energy from the phosphate group on a
free/new nucleotide is used to join it to the growing nascent DNA strand. If a mismatched nucleotide was
added, it can be removed and the energy from the correct free/new nucleotide can then be used to join it to
the strand. If polymerase instead synthesized in the 3’ to 5’ direction, then the energy would be derived from
the nucleotide on the existing strand, thus if an error occurred, a new correct nucleotide cannot provide
energy for its addition, causing inefficiency.
D1.1.7—Differences between replication on the leading strand and the lagging strand
Include the terms “continuous”, “discontinuous” and “Okazaki fragments”. Students should know
that replication has to be initiated with RNA primer only once on the leading strand but repeatedly
on the lagging strand.
Due to the antiparallel nature of DNA and directionality of DNA polymerase III, one strand will be
synthesized continuously towards the replication fork, and the other complementary parent strand will be
synthesized in fragments (called Okazaki fragments) away from the fork. This means that replication has
to be initiated with RNA primer only once on the leading strand but repeatedly on the lagging strand.D1.1.8—Functions of DNA primase, DNA polymerase I, DNA polymerase III and DNA ligase in
replication
Limit to the prokaryotic system.
The prokaryotic system for DNA replication involves several enzymes:
• DNA primase: adds RNA primers (segments of RNA ~5-10 nucleotides in length) complementary
to the parent strand to provide a 3’ OH group that allows DNA polymerase III to start synthesizing the
new strand.
• DNA polymerase III: adds nucleotides to the 3’ end of primers.
• DNA polymerase I: removes RNA primers and replaces them with DNA bases>
• DNA ligase: seals gaps between Okazaki fragments by forming phosphodiester bonds between
adjacent nucleotides to form one continuous DNA strand.
D1.1.9—DNA proofreading
Limit to the action of DNA polymerase III in removing any nucleotide from the 3' terminal with a
mismatched base, followed by replacement with a correctly matched nucleotide.
Errors in DNA replication are mitigated through DNA proofreading, which is a mechanism by which DNA
polymerase III immediately reads every new nucleotide after adding it to the growing DNA strand and
checks whether it is correct or not. The enzyme removes any nucleotide from the 3’ terminal with a
mismatched base and replaces it with a correctly matched nucleotide.
Linking questions
• How is genetic continuity ensured between generations?
• What biological mechanisms rely on directionality?
Review questions
SL and HL
• Suggest how the discovery of DNA’s helical structure immediately provided an idea as to how
DNA is passed to daughter cells. [1]
• State the role of the origin of replication. [1]
• The semi-conservative process of DNA replication is widespread across the domains of life.
Suggest how this feature of heredity contributes to genetic stability. [1]
• A research team observes that a particular strain of bacteria exhibits a high frequency of
mutations after UV exposure, specifically in regions where DNA is normally synthesized
discontinuously. Suggest which enzyme involved in DNA replication might be defective. [2]
• Evaluate how a mutation that increases the error rate of DNA polymerase could affect the
evolution of a rapidly reproducing virus. [2]
• Explain the role of short tandem repeats in biotechnology. [3]
• Explain the role of enzymes in DNA replication. [4]
• Discuss how knowledge of DNA replication enabled applications in biotechnology. [7]
• Discuss how a DNA sample is amplified and then separated in a paternity test. [8]Additional higher level
• A cell strain is isolated and it is discovered that the joining of Okazaki fragments is impaired due
to a mutation to an enzyme found at the replication fork. Suggest the enzyme most likely to be
mutated. [1]
• Outline how genetic errors are mitigated by the cell. [2]
• Explain the directionality of DNA polymerases. [3]
• Explain how genetic material is passed onto daughter cells in prokaryotic organisms. [7]References
Ann Clark, Mary, et al. Biology 2e. E-book, OpenStax, 2018, https://openstax.org/books/biology-2e/pages/1-introduction.
OpenStax.
Fan, Hao, and Jia-You Chu. “A brief review of short tandem repeat mutation.” Genomics, proteomics & bioinformatics vol.
5,1 (2007): 7-14. doi:10.1016/S1672-0229(07)60009-6.
Gordon Betts, J., et al. Anatomy and Physiology 2e. E-book, OpenStax, 2022, https://openstax.org/books/anatomy-and-
physiology-2e/pages/1-introduction. OpenStax.
Norris, Vic. “Does the Semiconservative Nature of DNA Replication Facilitate Coherent Phenotypic Diversity?.” Journal of
bacteriology vol. 201,12 e00119-19. 22 May. 2019, doi:10.1128/JB.00119-19.
Rogers, Kara. "gel electrophoresis". Encyclopedia Britannica, 11 Jun. 2024, https://www.britannica.com/science/gel-
electrophoresis.
Sholtis, S. (n.d.). Penn State DNA ladders: inexpensive molecular rulers for DNA research. Penn State University.
Yeah, I got you! This is a lot of info, but we can break it down into digestible parts. I’ll explain everything clearly and concisely, and we can go over anything you find confusing. Here’s a simple breakdown of the main topics:
1. DNA Replication Basics
• What? Copying DNA exactly before cell division.
• Why? Needed for reproduction, growth, and tissue repair.
• How? The double helix unwinds, and each strand serves as a template for a new complementary strand.
2. Semi-Conservative Replication & Base Pairing
• Semi-conservative = Each new DNA molecule has one old (parent) strand and one new strand.
• Complementary Base Pairing = A always pairs with T, and C always pairs with G. This prevents mistakes.
3. Enzymes in DNA Replication
• Helicase: Unzips the DNA by breaking hydrogen bonds.
• DNA Polymerase: Adds new nucleotides to form the new strand.
• DNA Primase: Adds RNA primers to start replication.
• DNA Polymerase I: Replaces RNA primers with DNA.
• DNA Ligase: Seals gaps (especially in the lagging strand).
4. Polymerase Chain Reaction (PCR)
• Purpose: Amplifies (makes many copies of) DNA.
• Steps:
1. Denaturation: Heat separates DNA strands.
2. Annealing: Cool it down so primers can attach.
3. Extension: Taq polymerase adds nucleotides.
• Why Taq Polymerase? It withstands high temperatures.
5. Gel Electrophoresis
• Purpose: Separates DNA fragments by size.
• Process:
• DNA is loaded into a gel.
• An electric current pulls DNA toward the positive side.
• Smaller fragments move further.
• A “DNA ladder” is used as a size reference.
6. Applications of PCR & Gel Electrophoresis
• DNA Profiling: Used in paternity tests and crime investigations.
• Short Tandem Repeats (STRs): Repeating DNA sequences unique to individuals, useful for matching DNA samples.
7. Directionality of DNA Polymerase
• 5’ to 3’ Direction: DNA polymerase can only add nucleotides in this direction.
• Leading Strand: Made continuously.
• Lagging Strand: Made in short Okazaki fragments.
8. DNA Proofreading
• DNA Polymerase III checks for mistakes and corrects errors.