DNA replication

What is DNA replication?

DNA replication is the production of an exact copy of DNA with an identical base sequence.

Why must DNA replicate?

DNA must replicate before cell division so that each daughter cell receives a complete copy of the genome. It is necessary for growth, repair and reproduction.

What is semi-conservative DNA replication?

Each new DNA molecule contains one original (parental) strand and one newly synthesised strand.

Why is DNA replication described as semi-conservative?

Because one strand of each original DNA molecule is conserved in each of the two new DNA molecules.

What is complementary base pairing?

Bases pair specifically: adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).

What bonds hold the two DNA strands together?

Hydrogen bonds between complementary bases hold the two DNA strands together.

How many hydrogen bonds form between A and T?

Adenine and thymine form 2 hydrogen bonds.

How many hydrogen bonds form between C and G?

Cytosine and guanine form 3 hydrogen bonds.

Why is complementary base pairing important in DNA replication?

It ensures that the sequence of bases on each original strand determines the exact sequence of the new complementary strand, allowing accurate copying of genetic information.

What is the first major step of DNA replication?

The DNA double helix is unwound and the two strands are separated, creating a replication fork.

What enzyme unwinds DNA during replication?

Helicase.

What does helicase do?

Helicase unwinds the DNA double helix and separates the two strands by breaking the hydrogen bonds between complementary bases.

What is a replication fork?

The region where the DNA double helix has been unwound and the two strands are separated so that new DNA strands can be synthesised.

What enzyme builds the new DNA strand?

DNA polymerase.

What does DNA polymerase do?

DNA polymerase links nucleotides together to form a new DNA strand using an existing DNA strand as a template.

How does DNA polymerase know which nucleotide to add?

It uses complementary base pairing: A pairs with T and C pairs with G.

Where does DNA polymerase add a new nucleotide?

DNA polymerase adds the new nucleotide to the 3′ carbon of the previous nucleotide.

What type of bond forms between nucleotides in the DNA backbone?

Covalent phosphodiester bonds form between the phosphate group of one nucleotide and the sugar of the next nucleotide.

What forms the DNA sugar-phosphate backbone?

Covalent phosphodiester bonds link the sugar and phosphate groups of adjacent nucleotides, forming a continuous sugar-phosphate backbone.

What is the role of primase in DNA replication?

Primase builds short RNA sequences called primers that provide a starting point for DNA polymerase.

What is a primer?

A short RNA sequence produced by primase that provides a starting point for DNA polymerase to begin DNA synthesis.

What are the two main enzymes involved in DNA replication?

Helicase and DNA polymerase.

What is the role of helicase compared with DNA polymerase?

Helicase separates the DNA strands by breaking hydrogen bonds, while DNA polymerase builds the new DNA strand by adding nucleotides.

Why does DNA replication produce genetically identical copies?

Complementary base pairing allows each original strand to act as a template, determining the exact sequence of the newly synthesised strand.

What happens to the original DNA strands during semi-conservative replication?

The two original strands separate, and each acts as a template for the formation of a new complementary strand.

What are the three main stages of PCR?

Denaturation, annealing, and DNA synthesis/extension.

What does PCR stand for?

Polymerase Chain Reaction.

What is the purpose of PCR?

PCR is used to amplify a specific region of DNA, producing many copies of the target DNA sequence.

What does it mean to amplify DNA?

To make many copies of a particular DNA sequence.

What is needed for PCR?

A DNA template, primers, heat-resistant Taq polymerase, free nucleotides and suitable conditions for DNA synthesis.

What is the DNA template in PCR?

The original DNA sample containing the region that needs to be amplified.

What are primers used for in PCR?

Primers are short DNA sequences that bind to specific complementary sequences and determine the region of DNA that will be replicated.

Why are two primers used in PCR?

One primer binds to each DNA strand, allowing DNA synthesis in both directions around the target region.

What enzyme is used in PCR?

Taq polymerase, a heat-resistant DNA polymerase.

What is Taq polymerase?

A heat-resistant DNA polymerase originally obtained from the bacterium Thermus aquaticus.

Why is Taq polymerase used in PCR?

It can tolerate the high temperatures used to separate DNA strands during PCR without being denatured.

What happens during PCR denaturation?

The DNA is heated to approximately 90°C, causing the double-stranded DNA to separate into two single strands.

Why is denaturation necessary in PCR?

The DNA strands must separate so that each single strand can act as a template for DNA synthesis.

What happens during PCR annealing?

The DNA is cooled to approximately 50°C, allowing the designed primers to attach to complementary sequences on the single DNA strands.

Why are primers important during annealing?

They identify the specific region of DNA to be amplified and provide a starting point for DNA polymerase.

What happens during PCR DNA synthesis/extension?

At approximately 70°C, Taq polymerase adds complementary nucleotides to the primers and forms new DNA strands.

What is the result of one PCR cycle?

The target DNA sequence is copied, and repeated cycles produce an exponential increase in the number of copies.

What are the three PCR stages in order?

1. Denaturation → 2. Annealing → 3. DNA synthesis/extension.

Why does PCR require repeated cycles?

Repeated cycles of denaturation, annealing and extension produce many copies of the target DNA region.

What is gel electrophoresis?

A technique used to separate DNA fragments according to their length.

What is the basic purpose of gel electrophoresis?

To separate DNA fragments so that their sizes can be compared and patterns can be analysed.

How does gel electrophoresis separate DNA fragments?

DNA samples are placed into wells in a gel and an electric current is applied. DNA fragments move through the gel at different rates depending on their length.

Why does DNA move during gel electrophoresis?

DNA has an overall negative charge, so it is attracted toward the positive electrode (anode) when an electric field is applied.

Which DNA fragments move fastest through the gel?

Smaller DNA fragments move through the gel more easily and therefore travel farther than larger fragments.

Which DNA fragments travel the shortest distance?

Larger DNA fragments move more slowly through the gel and therefore do not travel as far.

What is a DNA ladder in gel electrophoresis?

A set of DNA fragments of known lengths used as a reference for estimating the sizes of unknown DNA fragments.

What produces bands in gel electrophoresis?

DNA fragments of similar length group together, producing visible bands after the DNA is detected.

What determines the position of a DNA band in gel electrophoresis?

The length of the DNA fragments. Smaller fragments travel farther through the gel than larger fragments.

How is PCR used in forensics?

A small DNA sample from a crime scene can be amplified using PCR so there is enough DNA for DNA profiling.

How is gel electrophoresis used in forensics?

It separates DNA fragments according to length, allowing DNA profiles from different samples to be compared.

What is DNA profiling?

A technique used to produce a pattern of DNA fragments that can be compared between samples.

What are short tandem repeats (STRs)?

Short DNA sequences that are repeated multiple times in a person's DNA. Variation in the number of repeats can produce different DNA profiles.

How can restriction enzymes be used in DNA profiling?

Restriction enzymes act as molecular scissors, cutting DNA at specific base sequences to produce DNA fragments with different lengths.

How are PCR and gel electrophoresis used together in DNA profiling?

PCR amplifies selected DNA regions, restriction enzymes may cut DNA at specific sites, and gel electrophoresis separates the resulting fragments according to length to produce a profile.

What are applications of PCR?

PCR can be used to amplify DNA for applications such as forensic DNA profiling and other situations where only a small amount of DNA is available.

What are applications of gel electrophoresis?

It can be used to separate and analyse DNA fragments, including in DNA profiling and forensic investigations.

Why is PCR useful when only a small DNA sample is available?

PCR can amplify the target DNA region, producing many copies from a very small starting sample.

How is reliability improved in DNA profiling?

Using a greater number of DNA markers reduces the probability of obtaining a false match.

Why are multiple DNA markers used in DNA profiling?

Different individuals are unlikely to have the same combination of DNA characteristics across many markers, so using more markers reduces the probability of a false match.

What is the overall sequence of DNA replication?

Helicase separates the DNA strands → each strand acts as a template → complementary nucleotides are added by DNA polymerase → phosphodiester bonds form → two semi-conservative DNA molecules are produced.

What is the difference between DNA replication and PCR?

DNA replication occurs naturally in cells and copies the genome, while PCR is a laboratory technique used to amplify a specific DNA region.

What is the difference between DNA replication and gel electrophoresis?

DNA replication produces new DNA molecules, while gel electrophoresis separates existing DNA fragments according to length.

What are the key enzymes you need to know for D1.1 DNA replication?

Helicase separates the DNA strands; DNA polymerase adds nucleotides to build the new DNA strand; primase produces RNA primers. PCR uses the heat-resistant DNA polymerase Taq polymerase.

What is the directionality of DNA polymerase?

DNA polymerase can only add new nucleotides to the 3′ end of an existing DNA strand, so the new DNA strand is always synthesized in the 5′ → 3′ direction.

Why can DNA polymerase only add nucleotides to the 3′ end?

DNA polymerase requires an existing strand to add nucleotides to, and new nucleotides are added to the 3′ end. Therefore, DNA synthesis always proceeds 5′ → 3′.

In which direction is the new DNA strand synthesized?

5′ → 3′.

In which direction is the template strand read by DNA polymerase?

3′ → 5′, allowing the new complementary strand to be synthesized 5′ → 3′.

Why does DNA replication require different mechanisms on the two strands?

DNA is antiparallel and DNA polymerase can only synthesize DNA in the 5′ → 3′ direction, so the two strands must be replicated differently.

What is the leading strand?

The strand that can be synthesized continuously by DNA polymerase III as the replication fork opens.

How is the leading strand replicated?

It is synthesized continuously in the 5′ → 3′ direction by DNA polymerase III, following the movement of the replication fork.

What is the lagging strand?

The strand that must be synthesized discontinuously because DNA polymerase can only build DNA in the 5′ → 3′ direction.

How is the lagging strand replicated?

DNA polymerase III builds the new DNA in short sections called Okazaki fragments, which are later joined together.

What are Okazaki fragments?

Short sections of newly synthesized DNA formed discontinuously on the lagging strand.

Why are Okazaki fragments formed?

Because DNA polymerase can only synthesize DNA in the 5′ → 3′ direction while the DNA strands are antiparallel.

What is the main difference between leading and lagging strand replication?

The leading strand is synthesized continuously, whereas the lagging strand is synthesized discontinuously as Okazaki fragments.

Which enzyme synthesizes the new DNA on both the leading and lagging strands?

DNA polymerase III.

Which strand is synthesized continuously?

The leading strand.

Which strand is synthesized discontinuously?

The lagging strand.

What is the function of DNA primase?

DNA primase synthesizes short RNA primers that provide a starting point for DNA polymerase to begin DNA synthesis.

Why is a primer needed during DNA replication?

DNA polymerase cannot start a new DNA strand by itself; it can only add nucleotides to an existing strand, so a primer provides the starting point.

What does DNA polymerase III do?

DNA polymerase III adds nucleotides to the growing DNA strand in the 5′ → 3′ direction and forms phosphodiester bonds between nucleotides.

What does DNA polymerase I do?

DNA polymerase I removes the RNA primers and replaces them with DNA nucleotides.

What does DNA ligase do?

DNA ligase seals the gaps between adjacent DNA fragments, including Okazaki fragments, by forming phosphodiester bonds.

What enzyme joins Okazaki fragments together?

DNA ligase.

What enzyme replaces RNA primers with DNA?

DNA polymerase I.

What enzyme adds new DNA nucleotides to the growing strand?

DNA polymerase III.

What enzyme makes the RNA primers needed to start DNA synthesis?

DNA primase.

What is the correct order of the main enzymes involved in replication?

Primase provides the RNA primer → DNA polymerase III adds DNA nucleotides → DNA polymerase I replaces RNA primers with DNA → DNA ligase joins adjacent DNA fragments.

What is DNA proofreading?

A correction process during DNA replication in which errors in newly synthesized DNA are detected and corrected.

Why is DNA proofreading necessary?

DNA replication can produce incorrect base pairings, so proofreading helps detect and correct errors and maintain the accuracy of the DNA sequence.

Which enzyme carries out DNA proofreading according to the HL notes?

DNA polymerase III.

What is the role of DNA polymerase III in proofreading?

It not only adds new nucleotides during DNA replication but also detects and corrects many errors in the newly synthesized DNA.

Approximately how often does an incorrect base occur during replication before proofreading?

Approximately one incorrect base per 100,000 bases.

What happens to most errors made during DNA replication?

Most errors are detected and corrected through mechanisms such as proofreading.

Compare leading and lagging strand replication.

Both are synthesized by DNA polymerase III in the 5′ → 3′ direction using complementary base pairing. The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously as Okazaki fragments.