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[h] IB DP Biology HL D1.1 DNA replication Flashcards
[q] D1.1.1— What is DNA replication?
Why is it necessary?
[a] DNA replication the copying of DNA;
it is required for reproduction in all organisms;
and for growth;
and tissue replacement in multicellular organisms;
[q] D1.1.2—What does semi-conservative mean in terms of copying DNA?
Why does this lead to high levels of accuracy?
[a] Semi-conservative means that one strand of DNA is ‘old’ and one strand is newly made;
the double helix is opened up and both parent strands are used as templates for copying;
DNA is unwound;
new nucleotides are attached to the template strands;
according to complementary base pairing;
where adenine bonds with thymine and cytosine with guanine;
this means the copy is exact;
allowing a high degree of accuracy in copying base sequences.
[q] D1.1.3—What is the role of helicase in DNA replication?
[a] Helicase is responsible for unwinding of the double helix;
and separation of strands;
by breaking hydrogen bonds between two strands;
[q] D1.1.3 – What is the role of DNA polymerase (III)?
[a] DNA polymerase (III) attaches at the primer:
DNA polymerase III copies DNA;
by adding the new DNA nucleotides; to the template strands;
according to complementary base pairing;
using hydrogen bonds;
adenine pairs with thymine and cytosine pairs with guanine (*NOT A and T and G and C);
[q] D1.1.4—What is the polymerase chain reaction (PCR)?
What reactants are required?
What are the major steps?
[a] PCR is used to make very large number of copies of selected DNA sequences;
Uses Taq polymerase enzyme; DNA nucleotides, primers and a section of target DNA to copy;
DNA is heated to break hydrogen bonds between strands;
the mixture is cooled;
to allow short strands of DNA, called primers, to anneal (join to) or hydrogen bond to the target sequence;
Taq polymerase copies the strand; by adding in new DNA nucleotides according to complementary base pairing;
the cycle repeats; until there are huge amounts of DNA;
[q] D1.1.4— How does gel electrophoresis work?
On what basis are molecules separated?
[a] An electric current runs through a solution;
causing charged molecules to move through a gel;
DNA moves to the positive terminal, as it is negatively charged;
molecules are separated based on size;
the larger the molecule, the slower it moves;
the smaller, the faster and the further it moves through the gel;
[q] D1.1.5—What are the applications of polymerase chain reaction and gel electrophoresis?
[a] for e.g. DNA profiling for paternity case (to work out who the father is);
if there are bands that match between parent and child, they are related;
genetic engineering;
crime scene analysis;
where all bands must match;
Nature of Science: 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;
[q] AHL Only – D1.1.6— How are polymerases directional?
[a] DNA polymerases always add new nucleotides to the 3′ end of a of a DNA strand;
the 3′ end is the carbon on the bottom left of the sugar deoxyribose;
this is where a new sugar-phosphate bond is made;
[q] AHL Only – D1.1.7—What is the leading strand?
What is the lagging strand?
[a] Because new DNA nucleotides can only be added at the 3′ end of a DNA strand;
one strand can be synthesised continuously;
this is called the leading strand;
DNA polymerase binds at an RNA primer;
(and keeps going until the DNA is copied;)
whereas the other strand, the lagging strand,
DNA is synthesised discontinuously (not in one go);
in small sections called “Okazaki fragments”;
each section requires a new RNA primer;
[q] AHL Only – D1.1.8—What are the functions of DNA primase, DNA polymerase I, DNA polymerase III and DNA ligase in replication?
[a] DNA primase adds an RNA primer on parental DNA (both leading and lagging strands);
Which allows attachment of DNA polymerase III at the primer:
DNA polymerase III copies DNA in a 5´ (prime) → 3´(prime) direction (both leading and lagging);
by adding the new DNA nucleotides to the 3′ end; of the template strands;
through hydrogen bonding;
according to complementary base pairing;
adenine pairs with thymine and cytosine pairs with guanine (*NOT A and T and G and C);
DNA polymerase I removes the RNA primers on the lagging strand;
and replaces them with DNA;
DNA ligase joins Okazaki fragments;
by forming covalent sugar-phosphate bonds;
[q] AHL Only – D1.1.9—DNA proofreading
[a] DNA Polymerase III also proof-reads to check for errors;
removing any nucleotide from the 3′ terminal with a mismatched base;
followed by replacement with a correctly matched nucleotide;
[q] DNA replication
[a] Production of exact copies of DNA with identical base sequences
[q] DNA replication role in multicellular organisms
[a] Reproduction: gametes pass on genetic information
Cell division: generate new cells for growth and tissue repair
[q] Semi-conservative nature of DNA replication
[a] The original DNA is divided into two strands and used as template strands to form 2 new identical daughter DNA
[q] Helicase
[a] An enzyme that untwists the double helix of DNA at the replication forks.
[q] Directionality of DNA polymerase
[a] New nucleotides are added to the 3′ hydroxyl (-OH) group of the previous nucleotide, forming a phosphodiester bond and extending the chain in a 5′ to 3′ direction.
[q] DNA polymerase I
[a] Removes RNA nucleotides of primer from 5′ end and replaces them with DNA nucleotides
[q] DNA polymerase III
[a] adding bases to the new DNA chain; proofreading the chain for mistakes
[q] DNA primase
[a] synthesis of RNA primer
[q] Leading strand
[a] The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5′ to 3′ direction.
[q] Lagging strand
[a] A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5′ to 3′ direction away from the replication fork.
[q] Okazaki fragments
[a] Small fragments of DNA produced on the lagging strand during DNA replication
[q] DNA ligase
[a] Bind Okazaki fragments together
[q] DNA replication steps
[a] 1) Helicase unwinds the parental double helix
2) Lagging strand: DNA Primase creates RNA primers for Okazaki fragments DNA polymerase III synthesized
Leading strand: DNA polymerase III continuously adds nucleotides to the leading strand as it unwinds
3) DNA polymerase I removes the RNA primers and replaces them with DNA nucleotides; DNA ligase joins Okazaki fragments together.
4) DNA replication is completed when the entire DNA molecule has been copied
[q] Polymerase chain reaction(PCR)
[a] process to amplify DNA a reaction used to make copies of segments of DNA(a particular gene)
[q] PCR steps
[a] 1. Denaturation: The double-stranded DNA template separate into single strands due to high temperature
2. Annealing: The temperature is then lowered to allow the primers to bind to the complementary single-stranded DNA templates
3.Extension: The temperature is raised again, Taq polymerase synthesizes new DNA strands by extending the primers, using the original DNA strand as a template.
4. Repetition of Step 1-3
[q] Use of primers in PCR
[a] Short strands of nucleotides which are complementary to specific target sequences at the two ends of the region of DNA to be amplified
[q] Temperature changes in PCR
[a] 1. HEATING to 95C: Breaks hydrogen bond to separate DNA strand
2. COOLING to 53C: Allows the primers to bind to their complementary base sequences, provide the starting point for DNA polymerase to add nucleotides
3. HEATING TO 73C: Allows Taq DNA polymerase to work at optimum temperature, extending the primers by adding new DNA nucleotides to the new strands
[q] Applications of PCR
[a] Allows the investigation of small samples of DNA
– Forensics: Amplifying DNA for genetic fingerprinting analysis
– Amplifying viral or bacterial DNA in patients’ blood when diagnosing disease
[q] Gel electrophoresis
[a] Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel
[q] Applications of Gel electrophoresis
[a] – creating DNA fingerprints
– analysis of genes associated with genetic illness
– determining paternity
– determining evolutionary relationships
[q] Parent DNA
[a] The original DNA that replicates
[q] Daughter DNA
[a] The two identical strands of DNA that are created as a result of DNA synthesis.
[q] semi-conservative replication
[a] in each new DNA double helix, one strand is from the original molecule, and one strand is new
[q] DNA replication
[a] The process in which DNA makes a duplicate copy of itself.
[q] Unwinding of DNA
[a] Enzyme unzips the 2 strands so that they separate- results in 2 single strands of DNA
[q] Synthesis of DNA
[a] loose nucleotides attached to separate strands of DNA via complementary base pairing.
[q] Ligation of DNA
[a] DNA synthesis on lagging strand continues until it reaches previously synthesized DNA
DNA polymerase I removes the RNA primer and replaces it with DNA
DNA ligase seals nicks in the DNA
[q] Helicase
[a] An enzyme that untwists the double helix at the replication forks, separating the two parental strands and making them available as template strands.
Breaks the Hydrogen bonds between nitrogen bases.
[q] Topisomerase
[a] works in front of helicase, helps prevent overwinding of DNA at the replication fork
[q] RNA primase
[a] An enzyme that creates an RNA primer for initiation of DNA replication.
[q] DNA polymerase III
[a] synthesizes new DNA only in the 5′ to 3′ direction
[q] DNA Polymerase I
[a] Removes RNA nucleotides of primer from 5′ end and replaces them with DNA nucleotides
[q] DNA ligase
[a] an enzyme that eventually joins the sugar-phosphate backbones of the Okazaki fragments
[q] replication fork
[a] A Y-shaped region on a replicating DNA molecule where new strands are growing.
[q] single strand binding proteins
[a] A protein that binds to the unpaired DNA strands during DNA replication, stabilizing them and holding them apart while they serve as templates for the synthesis of complementary strands of DNA.
[q] leading strand
[a] The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5′ to 3′ direction.
[q] lagging strand
[a] A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5′ to 3′ direction away from the replication fork.
[q] Okazaki fragments
[a] Small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete strand.
[q] 5′ to 3′
[a] direction of DNA replication
[q] What does DNA Ligase bond?
[a] 5′ phosphate and 3′ hydroxyl group
[q] Are daughter strands identical to the parent strand of DNA?
[a] yes
[q] Exon
[a] expressed sequence of DNA; codes for a protein
[q] Intron
[a] sequence of DNA that is not involved in coding for a protein
[q] What are some functions of noncoding regions (introns)?
[a] Acting as a guide for producing tRNA and rRNA
Regulating gene expression as enhancers or silencers
[q] Telomeres
[a] Repeated DNA sequences at the ends of eukaryotic chromosomes.
[q] gel electrophoresis
[a] Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel.
[q] DNA Fluorescent sequencing
[a] copies of DNA are placed in a test tube with replication enzymes.
small amounts of dideoxyribonucleotides are added that have been fluorescently tagged.
These lack a 3′ -OH group, so replication halts after they are incorporated.
The resulting strands are sorted using gel electrophoresis.
The sequence of bases can be determined by the fluorescent pattern.
[q] short tandem repeats (STRs)
[a] repeating sequences of noncoding DNA
[q] DNA profiling
[a] A procedure that analyzes DNA fragments to determine whether they come from a specific individual.
[q] What are uses for DNA Profiling?
[a] Establish paternal lineage by studying the Y-chromosome
Establish maternal lineage by studying mitochondrial DNA
Identify individuals by forensic testing.
[q] 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.
[a] 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
[q] 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.
[a] 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.
[q] 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.
[a] 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 ATP
[q] D1.1.4—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.
[a] 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. 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
2. Annealing: the sample is cooled in order to anneal (add) the RNA primers which are needed for initiating DNA polymerase activity
3. 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
[q] D1.1.4—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.
[a] 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).
[q] D1.1.4—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.
[a]
[q] 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.
[a] 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.
[q] 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.
[a] 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.
[q] 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.
[a] 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.
[q] 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.
[a] 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.
[q] D1.1.8—Functions of DNA primase, DNA polymerase I, DNA polymerase III and DNA ligase in replication
Limit to the prokaryotic system.
[a] 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.
[q] 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.
[a] 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.
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