IB BIOLOGY · THEME D · CONTINUITY AND CHANGE

Copied in full, every time a cell divides

Before a cell divides it copies all of its DNA, base by base, and gets almost every one right. Here is how two enzymes unzip and rebuild the helix, how a lab copies a speck of DNA a billion times, and how a profile can name a parent or a suspect.

IB Biology D1.1SL and HL2 hours SL, 4 HL10 chaptersdry lab

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One moleculeUnzippedEach half rebuiltTwo copiesGHCHCHGHTHAHAHTHAHTHTHAHCHGHGHCHGHCHCHGHAHTHTHAHTHAHAHTHCHGHGHCH
One round of copying: unzipped from the top, each half rebuilt, two molecules that each keep one old strand.

01Learn

Ten chapters, the last four for higher level, and a game to play twice. Read a chapter, step or tap its figure, and make each prediction before you open the answer.

  1. 1A copy for every new cell
  2. 2One old strand, one new
  3. 3Unzip, then build
  4. 4A millionfold copy
  5. 5Sorted by size
  6. 6Profiles, parents and crime scenes
  7. 7Only ever at the 3′ end HL
  8. 8One strand smooth, one in pieces HL
  9. 9Four enzymes at the fork HL
  10. 10Checking as it goes HL

D1.1.1 Exact copies

A copy for every new cell

Almost every cell in your body came from another cell dividing. Before a cell divides it has to hand each of the two new cells a full set of instructions, and the instructions are written in its DNA. Making a copy of a DNA molecule is called DNA replication.

The copy has to be exact. The message in DNA is the order of its bases, the base sequence (A1.2), so a faithful copy has the same bases in the same order. Change the sequence and you may change a protein. A change that slips through is a mutation (D1.3), which is why copying has to be so careful. Chapter 2 shows how the pairing rule keeps it accurate, and chapter 10 how the enzyme checks its own work.

Copying comes before every cell division. In a eukaryote it happens in the part of interphase before mitosis, when each chromosome’s DNA molecule becomes two identical molecules (D2.1 has the cell cycle). Cells divide for three reasons.

  • Reproduction. A bacterium splits in two, yeast buds and a strawberry plant sends out runners: asexual reproduction. Even the cells that become eggs and sperm come from cells that copied their DNA first.
  • Growth. A fertilised egg becomes a body of about 30 trillion cells, every one made by division. This is growth.
  • Tissue replacement. Skin, the lining of the gut and blood cells are replaced all the time, and a cut heals with new cells: tissue replacement.

Copying is how genetic continuity runs from one cell to the next, and from parent to offspring.

Watch out Replication is not the same as cell division. Replication copies the DNA; the cell divides afterwards. And the copy is of the whole molecule, not of one gene.

You began as one fertilised egg and now have about 30 trillion cells. If every cell divided in step, about how many rounds of copying and dividing would it take?

one cell, one copy of its DNAcopieddividedcopy, then dividereproductiongrowthnewtissue replacement1, 2, 4, 8, 16 ...30 trillion?every cell made by division
  1. A cell holds one copy of its DNA.
  2. Before it divides, it copies it, so each new cell has a full set.
  3. Cells divide to reproduce, to grow and to replace what is lost.
  4. Every one of your cells came from one by division.

D1.1.2 Semi-conservative replication

One old strand, one new

The two strands of a double helix are held together by hydrogen bonds between their bases (A1.2). To copy the molecule, the cell pulls the strands apart. Each old strand then acts as a template: free nucleotides line up against its exposed bases, A opposite T and C opposite G, and are joined into a new strand. That is complementary base pairing, and nothing else fits.

So each new molecule, a daughter molecule, is made of one old strand and one new one. That is what semi-conservative means: half of each molecule is conserved from the one before.

Why is the copy so accurate? The pairing rule allows only one base opposite each base of the template. A new strand is therefore the exact complement of its template, and the complement of a complement is the original, so both daughter molecules carry the base sequence of the molecule that went in. Accurate is not the same as perfect: very rarely a wrong base is joined in, and chapter 10 shows how the enzyme catches it.

  • Each old strand is a template for one new strand.
  • Base pairing, A with T and C with G, makes each new strand the complement of its template.
  • Each daughter molecule: one whole old strand and one whole new strand.

A DNA molecule is copied, and then both copies are copied again. Of the four molecules, how many still contain a strand from the original?

Watch out Semi-conservative does not mean each new strand is half old and half new. Every strand is entirely old or entirely new; it is the molecule that is half and half.
How we know (beyond the IB guide’s statements)

People once proposed two other patterns: both old strands staying together in one molecule (conservative), or old and new pieces mixed along every strand (dispersive). An experiment decided between the three.

In 1958 Matthew Meselson and Franklin Stahl grew the bacterium E. coli for many generations on food whose nitrogen was the heavy isotope nitrogen-15, so all of its DNA was heavy. They moved the bacteria to food with ordinary nitrogen-14 and took samples after one and after two generations.

Spun very fast in a dense salt solution, DNA settles as a band at the height that matches its density: heavy low, light high, hybrid in between. Each model predicts a different pattern, so the results could rule two of them out. Choose a model to see the tubes it predicts, then what they saw.

lighthybridheavystartafter 1generationafter 2generationspredictedpredictedpredictedpredictedpredictedpredictedpredictedpredictedpredictedobservedobservedobservedthicker band, more DNA
Each tube spins DNA into bands by density. Pick a model, then compare it with what was seen.
GHTHoldCHAHoldCHAHGHTHCHAHnewGHTHnewtemplatetemplateGHTHoldCHAHCHAHnewtemplateCHAHnewGHTHGHTHnewtemplateCHAHoldGHTHGHTHnewtemplateGHTHnewCHAHCHAHnewtemplate
  1. One molecule: two old strands.
  2. A new partner is built along each strand: its template.
  3. Each new molecule keeps one old strand: semi-conservative.
  4. Copied again: every strand is a template.
  5. Count the old strands.

D1.1.3 Helicase and DNA polymerase

Unzip, then build

Copying is two jobs, and a different enzyme does each. First the helix has to open, because inside it every base is already paired and tucked away. Then a new strand has to be built against each old one.

Helicase is the unzipper. It moves along the double helix, unwinding it and breaking the hydrogen bonds between the base pairs, so that the two strands part. The point where the helix is opening is the replication fork. Only hydrogen bonds break. The covalent bonds along each strand stay whole, so each template keeps its sequence.

DNA polymerase is the builder. Free nucleotides drift in the nucleus, or in the cytoplasm of a bacterium. One whose base pairs with an exposed base on the template is joined to the end of the new strand by a covalent bond in the sugar and phosphate backbone. DNA polymerase only joins a nucleotide that pairs correctly, which is why the copy is accurate. It adds whole nucleotides, already made: it does not make bases.

Each template strand has its own DNA polymerase. The strands run in opposite directions, so the two enzymes work in opposite directions, and that makes the two new strands very different: chapter 8 explains how.

  • Helicase unwinds the helix and breaks the hydrogen bonds between bases.
  • DNA polymerase joins free nucleotides into a new strand, matched to the template by base pairing.
  • One polymerase works on each template strand.

That answers the first guiding question. New DNA is made by unzipping the old and building a complementary strand on each half. Next, do it yourself: Run the fork.

Watch out Helicase breaks the hydrogen bonds between the bases, not the bonds between sugar and phosphate. And DNA polymerase does not “make bases”: it joins whole nucleotides, already made, into the new strand.

A drug stops helicase but leaves DNA polymerase working. What happens to replication?

GHAHTHCHAHTHAHCHGHGHCHTHAHGHGHAHTHGHCHCHAHGHTHGHAHTHCHCH5′3′5′3′3′5′5′3′helicaseDNA polymeraseDNA polymerasehydrogenbonds

Tap a part of the fork.

Old strands are on pink bands, new strands on green bands with a dashed edge. The two polymerases face opposite ways because the strands do.
GHCHCHGHTHAHAHTHhelicaseoldoldhelicaseoldoldnew??DNApolymeraseDNApolymeraseoldnewnewold
  1. One molecule: two old strands.
  2. Helicase unwinds the helix and breaks the hydrogen bonds.
  3. DNA polymerase joins nucleotides that pair with each exposed base.
  4. Two molecules, each with one old strand and one new.

D1.1.4 The polymerase chain reaction

A millionfold copy

A smear of blood, a hair root or a swab holds far too little DNA to study. The polymerase chain reaction (PCR) copies one chosen stretch of DNA over and over until there is plenty. Making many copies of a small amount of DNA is to amplify it.

The tube holds the sample, plenty of free nucleotides, a buffer, a polymerase and two primers: short single strands of DNA, one matching each end of the stretch to be copied. A thermal cycler then changes the temperature again and again. One cycle has three steps.

  • Denaturation, about 95 °C. Heat breaks the hydrogen bonds and the strands separate. It does helicase’s job.
  • Annealing, about 55 °C. The mixture cools and a primer binds, by hydrogen bonds, to its matching sequence at each end of the target.
  • Extension, about 72 °C. The polymerase adds nucleotides onto each primer and builds a complementary strand.

A cycle takes two or three minutes, and 30 cycles take an hour or two. Each cycle doubles the number of copies, so after n cycles one molecule can give up to 2n: 30 cycles give 1,073,741,824. In practice the doubling slows near the end, as primers and nucleotides run low.

Doubling A crime-scene swab holds about 10 copies of the stretch to be tested. After 30 cycles there are 10 × 230 = 10 × 1,073,741,824, about 10.7 billion copies: enough to see as a band on a gel (chapter 5).

Why primers? A DNA polymerase cannot start a strand on its own. It can only add to the end of a strand that is already paired with the template. The primers give it that start, and their sequences choose which stretch gets copied; chapter 8 shows that a cell has the same problem.

The polymerase used is Taq polymerase, from Thermus aquaticus, a bacterium found in hot springs.

PCR mimics replication: strands are separated, nucleotides pair with a template and a polymerase joins them. What differs is that heat does the unzipping instead of helicase, the primers are made by a chemist, and one short stretch is copied instead of a whole chromosome. Chapter 5 shows how the copies are then sorted by size.

Remember it One PCR cycle

  • DragonsDenaturationabout 95 °C: the strands separate
  • AlwaysAnnealingabout 55 °C: primers bind
  • ExhaleExtensionabout 72 °C: Taq builds the new strands
Watch out PCR does not copy a whole genome, and it does not make a new organism. It copies one short stretch chosen by the primers. And denaturing DNA is harmless to it: the strands come apart and pair again, unlike a protein that loses its shape for good.

PCR uses a DNA polymerase from bacteria that live in hot springs. Why not a polymerase from human cells?

targetflanking DNAprimerprimerTaq polymerase (the green clamps)copies of the target012345cycles?301, 2, 4, 8, 16, 32 ...20406080100°Ctime25 °Cdenaturationannealingextensioncycle 2: fourcycle 1
  1. A sample of DNA, too little to study.
  2. About 95 °C: the strands separate.
  3. About 55 °C: primers bind at each end of the target.
  4. About 72 °C: Taq polymerase extends each primer.
  5. One cycle: the copies double.
  6. Thirty cycles later: how many?

D1.1.4 Gel electrophoresis

Sorted by size

PCR gives you a tube of identical copies. To see what is in it, you sort the DNA by size. Gel electrophoresis does that: it separates DNA fragments by their length, counted in base pairs (bp).

A slab of gel, a jelly made from seaweed, has a row of small pits at one end, the wells. The slab lies in a tank of salty buffer between two electrodes. Each sample is mixed with a dense loading solution, so it sinks into its well, and a blue dye in the mix shows how far the run has gone. Then the current goes on.

Why does DNA move? Every nucleotide carries a phosphate group with a negative charge, so DNA is pulled towards the positive electrode, and the wells go at the negative end. At the bench: run to red, because the positive lead is usually red.

Why does it separate? The gel is a mesh of fibres. Short fragments slip through quickly and long ones are held back more. Every fragment carries about the same charge for each base pair, so the pull on each base pair is the same, and size decides how far a fragment gets in a set time. The shortest travel furthest.

A DNA ladder, a mix of fragments of known sizes, runs in its own lane and gives a scale. To size an unknown band, find the ladder band level with it, or the two it sits between.

DNA is colourless, so afterwards the gel is soaked in a stain that glows under blue or ultraviolet light when it binds DNA. Each set of same-length fragments shows as one band, so the number of bands is the number of different fragment lengths in the sample, and the distances between bands show their relative sizes. A band holds millions of identical fragments, because a single molecule is far too little to see. That is why PCR comes first.

  • The negative charge of the phosphates pulls DNA towards the positive electrode.
  • The mesh holds long fragments back more, so short ones travel furthest.
  • The ladder gives the scale for reading sizes.

One more fact for the curious: distance travelled is not in proportion to size. It falls roughly with the logarithm of the size, so doubling a fragment’s length does not halve its distance. And real profiling labs run the same separation in a hair-thin tube of gel, a capillary, that can tell apart fragments one base pair different in length. Textbooks, and this guide, draw slab gels.

Watch out Bigger fragments do not travel further because they are “heavier” or “more charged”: they travel less far, because the gel holds them back. And DNA moves to the positive electrode, because it is negative.

Fragments of 300, 1,000 and 5,000 base pairs are run on the same gel. Which ends up nearest the wells?

−negative electrode+positive electrodeladdersample 1sample 2sample 3emptywellsblue dyebp1002003005001,0002,000

0

Seen from above. The DNA is stained, so every band shows. The sizes are in base pairs.

D1.1.5 Applications of PCR and gel electrophoresis

Profiles, parents and crime scenes

PCR and gel electrophoresis are used far beyond the classroom. A few of the jobs they do:

  • Forensic investigations and paternity tests, by DNA profiling.
  • Diagnosing infections. The COVID-19 test copies a stretch of the virus’s genes (the virus’s RNA is first copied into DNA).
  • Testing for inherited conditions.
  • Identifying species: a fish sold as cod that is not, the source of a meat sample, wildlife trading.
  • Copying a gene so that it can be studied or moved.
  • Studying ancient DNA from bones.

Most of our DNA is the same from one person to the next, but some places hold a short sequence repeated over and over, end to end: a short tandem repeat (STR), such as GATA GATA GATA. People differ in how many repeats they have. Such a place is a marker, each version (a number of repeats) is an allele, and each person has two alleles at each marker, one from each parent.

DNA profiling starts with a sample: blood, saliva, a hair root, or skin cells left on a handle. PCR copies the markers, using primers that bind either side of each repeat. Electrophoresis then separates the copies by length: more repeats, longer copy, shorter distance. The pattern of bands at several markers is the person’s profile.

In a forensic investigation, the profile from the scene is compared with those of the suspects and the victim. A match at every marker is strong evidence that the sample came from that person, and a single clear mismatch excludes them. Profiling has cleared innocent people as well as convicting guilty ones.

In a paternity test, a child has at each marker one allele from the mother and one from the father. A band in the child that the mother does not have must come from the father, so a man without it is excluded. Labs look for exclusions at two or more markers, because a rare new mutation can change one.

So how many markers are enough? One marker proves little, because many people share any one pair of alleles. Each extra independent marker is another test that a person must pass by luck, so the chance of a false match is multiplied by another small number and falls very fast. Reliability rises with the number of measurements. Real profiles use about 15 to 20 markers: 16 in the UK and 20 in the USA.

Take care all the same. Identical twins share a profile, close relatives share more than strangers, and a sample can be contaminated. The evidence is strong, but not proof on its own.

Watch out A child does not share half of each parent’s bands in some fixed way. At each marker the child has exactly one allele from each parent, and those may happen to be the same size as the other parent’s. And a profile is not the person’s whole genome: it is a handful of repeat counts.

At one marker, 1 person in 10 shares the crime-scene pattern by chance. With ten independent markers, each 1 in 10, how many people in a city of 10 million would you expect to match by chance?

repeats8910111213141516laddermotherchildman Aman Bladderscenevictimsuspect 1suspect 2suspect 3

One marker only. Would you stop here?

The ladder has a band for every allele, numbered by its repeats. More repeats make a longer copy, which travels less far.
1 marker: about 1 person in 10 matches by chance.In this city of a million, that is about 100,000 people.3 markers: about 1 person in 1,000 matches by chance.About 1,000 people in this city: just one dot.10 markers: about 1 person in 10 billion.Probably nobody in the city.20 markers: about 1 in 10²⁰.Far fewer than one person in everyone who has ever lived.a city of a million peoplematches by chanceeach dot is 1,000 people
Each marker is taken as shared by about 1 person in 10, so each extra marker divides the chance of a false match by 10. Real markers vary, and many are rarer.

D1.1.6 Directionality of DNA polymerases

Only ever at the 3′ end

From A1.2: the carbons of the sugar in a nucleotide are numbered 1′ to 5′, and in a strand each phosphate links the 5′ carbon of one nucleotide to the 3′ carbon of the one before. So the two ends of a strand are different. One end has a free phosphate on a 5′ carbon, the 5′ terminal. The other has a free hydroxyl group (OH) on a 3′ carbon, the 3′ terminal.

DNA polymerases follow one rule. They join the 5′ phosphate of an incoming nucleotide to the 3′ OH at the end of the strand they are building, by a covalent bond in the sugar and phosphate backbone. So nucleotides are only ever added at the 3′ end, and a new strand grows 5′ to 3′. This one-way working is the directionality of DNA polymerases.

The template is read the other way. As the new strand grows 5′ to 3′, the polymerase moves along its template 3′ to 5′, because the two strands are antiparallel. At a fork the two templates run in opposite directions, so the two new strands have to grow in opposite directions. Chapter 8 follows what that means.

A polymerase also cannot start a strand from nothing. It needs a 3′ end to add to, which is what the primers of chapter 8 are for.

Where the energy comes from (not needed for the exam) Free nucleotides arrive carrying three phosphates. DNA polymerase cuts off two, and the energy released makes the new bond.
  • Nucleotides are added only at the 3′ end of the new strand.
  • So a new strand grows 5′ to 3′, and its template is read 3′ to 5′.
  • Antiparallel templates mean the two new strands at a fork grow in opposite directions.

Directionality rules other copying too. RNA is built 5′ to 3′ in transcription, and a ribosome reads messenger RNA 5′ to 3′ in translation (D1.2).

Watch out “5′ to 3′” describes the new strand. The template is read 3′ to 5′. And 5′ and 3′ name carbons of the sugar, not ends of a gene.

A free nucleotide arrives at the 5′ end of a growing new strand, and its base pairs with the template. What happens?

AHGHCHTHAHCH5′3′AHTHAHTHGHAHTHGHAHTHCHGHAHTH3′3′3′5′GH5'3'5′ phosphateto 3′ OHGH5'3'no free 3′ OHhereCH5'3'template: read 3′ → 5′new strand: grows 5′ → 3′THCHGHAHTHGHAHGHCHTH3′5′5′3′two templates, two directions
  1. Every strand has a 5′ end and a 3′ end.
  2. DNA polymerase joins a nucleotide’s 5′ phosphate to the 3′ end.
  3. There is nothing to join to at the 5′ end.
  4. So new strands grow 5′ to 3′.
  5. The templates are antiparallel, so the new strands grow in opposite directions.

D1.1.7 Leading and lagging strands

One strand smooth, one in pieces

The fork moves along the DNA as helicase unwinds it. One template is the right way round for its new strand, growing 5′ to 3′, to grow in the same direction as the fork moves. DNA polymerase follows right behind helicase and builds without stopping. This is the leading strand, made continuously.

On the other template the new strand must also grow 5′ to 3′, and for this one that is away from the fork. As the fork opens a fresh stretch of template, a polymerase starts near the fork and builds back towards the piece before it. Then the fork opens a little more and the polymerase starts again. This is the lagging strand, made discontinuously in short pieces called Okazaki fragments.

A polymerase cannot start a strand from nothing, as chapter 7 said. So every new piece of DNA begins with a short RNA primer, made by an enzyme you meet in chapter 9, which gives the polymerase a 3′ end to add to. The leading strand needs a primer only once, at its start. The lagging strand needs one for every Okazaki fragment.

Why “lagging”? Its pieces are finished a little behind the fork. Its polymerase is not lazier: both strands are made at the same fork at the same time. The pace differs, though. The leading strand grows steadily, at the speed the fork opens; the lagging strand grows in stop-start bursts, one fragment at a time, and keeps up only on average.

In E. coli Okazaki fragments are about 1,000 to 2,000 nucleotides long, and the fork moves about 1,000 base pairs a second. Copying begins at one place on the circular chromosome and two forks travel away from it in opposite directions, meeting on the far side about 40 minutes later, for 4.6 million base pairs. Each fork has its own leading and lagging strand.
  • Leading strand: built continuously towards the fork, from one primer.
  • Lagging strand: built discontinuously, away from the fork, as Okazaki fragments, each from its own primer.
  • Both are made at the same fork at the same time.

You will run both strands yourself in Run the fork’s higher level rounds, after chapter 10.

Watch out The lagging strand is not copied later, after the leading strand is finished: both are made at the same fork at the same time. And “continuous” and “discontinuous” describe how each new strand is built, not the template.

A fork opens 10,000 bases of a bacterial chromosome. Okazaki fragments there are about 1,000 to 2,000 nucleotides long. How many RNA primers does each new strand need?

3′5′5′3′5′3′3′5′5′3′5′3′3′5′3′5′5′3′5′3′3′5′3′5′3′5′5′3′5′3′3′5′3′5′3′5′3′5′5′3′5′3′3′5′3′5′3′5′3′5′5′3′helicaseleading strand:continuouslagging strand:Okazaki fragments1 primer3 primers
  1. Helicase opens the fork and moves on.
  2. Leading strand: one primer, then built continuously, towards the fork.
  3. Lagging strand: built away from the fork, so it must start again.
  4. Each new stretch gets its own primer.
  5. The lagging strand comes in Okazaki fragments.
  6. One primer on the leading strand; one per fragment on the lagging strand.

D1.1.8 Primase, polymerases I and III, ligase

Four enzymes at the fork

Helicase has opened the fork. Four more enzymes finish the job. This is the system of a bacterium such as E. coli, which is the one the IB asks for, and the order they act in is the order to learn.

  • DNA primase makes a short RNA primer (about ten nucleotides in E. coli) on the template, complementary to it: once at the start of the leading strand, and once for each Okazaki fragment.
  • DNA polymerase III extends each primer from its 3′ end, one nucleotide at a time, each paired with the template, so it builds almost all of each new strand, 5′ to 3′. It also proofreads (chapter 10).
  • DNA polymerase I removes the RNA primers and replaces their nucleotides with DNA nucleotides. Like every DNA polymerase it needs a 3′ end to add to, so it works from the 3′ end of the newer fragment that has grown up to the primer, taking out RNA ahead of it and filling in DNA behind. A primer is only replaced once the next fragment has reached it.
  • DNA ligase joins the fragments. It makes the last covalent bond in the sugar and phosphate backbone, where one fragment’s 3′ end meets the next fragment’s 5′ end. The unjoined gap is a nick.

Why RNA primers, and then their removal? The finished strand must be DNA all through, so every primer’s RNA has to be swapped out. That is also why ligase works many times on the lagging strand, once at every join, and once on the leading strand, where its single primer was replaced. It also answers why neighbouring Okazaki fragments are not joined at first: between them lies an RNA primer at first, then, once polymerase I has swapped it for DNA, a nick, and only then can ligase seal it.

Helicase, from chapter 3, is the fifth enzyme at the fork: named again here, not taught again.

Remember it The enzymes at the fork, in order

  • HungryHelicaseunwinds and breaks hydrogen bonds
  • PandasDNA primaselays an RNA primer
  • PreferDNA polymerase IIIbuilds the new strand 5′ to 3′, and proofreads
  • PlainDNA polymerase Iswaps RNA primers for DNA
  • LettuceDNA ligaseseals the nicks
Watch out DNA polymerase III does not remove primers: polymerase I does. Ligase does not add nucleotides: it makes one bond where two pieces meet. And primers are RNA, which is why they must be replaced.

A bacterium makes a faulty DNA ligase. What would you find in its newly copied DNA?

DNA polymerase IIIhelicaseDNA primaseDNA polymerase IIIDNA polymerase IDNA ligase

Tap an enzyme to find out what it does.

The fork of the last figure at a later moment. Old strands are pink, new DNA is green and primers are hatched yellow.

D1.1.9 DNA proofreading

Checking as it goes

Base pairing makes the right base far likelier than a wrong one, but now and then DNA polymerase III joins a nucleotide whose base does not pair with the template. That is a mismatch.

Proofreading deals with it. DNA polymerase III checks the nucleotide it has just added. If its base is a mismatch, the enzyme backs up, removes that nucleotide from the 3′ terminal of the new strand, adds a correctly matched one in its place, and carries on. It is the last nucleotide that gets checked because a mismatched base at the 3′ end does not pair properly, and the polymerase cannot extend well from it. That is how the mistake is caught before it is buried.

How good is it? Without proofreading, DNA polymerase III gets about 1 base in 100,000 wrong. Proofreading catches about 99 in 100 of those, so about 1 in 10 million slips through. (Cells have further checks after copying, which take it lower still, but they are beyond the IB guide.)

A mismatch that is left in becomes a mutation in one of the two daughter molecules after the next copying (D1.3). Accuracy matters because every cell copies billions of bases.

  • A mismatch is a new nucleotide whose base does not pair with the template.
  • DNA polymerase III backs up, removes it from the 3′ end and adds the correct one.
  • It checks as it builds, not afterwards.

Accurate copying plus proofreading is how genetic continuity is kept between generations, and the rare errors that remain are where new alleles come from.

Now put the whole fork together in Run the fork, below.

Watch out Proofreading does not check the whole strand afterwards: it removes the nucleotide just added at the 3′ end, while the strand is being built. And it is DNA polymerase III that proofreads (in the IB’s account), not polymerase I or ligase.

Without proofreading, DNA polymerase III gets about 1 base in 100,000 wrong. With it, about 1 in 10 million. Copying a bacterial chromosome means adding about 9 million new bases. About how many mistakes would each give?

AHTHCHGHAHGHCHTHGHCHTHGHCHTHAHGHCHTHTHAHGHCHTHGHmismatcha G opposite a T:the edges do not fitremoved from the3′ endreplacedan A opposite a T:the edges fitchecked: every baseDNApolymerase III
  1. DNA polymerase III adds nucleotides at the 3′ end.
  2. Rarely, a mismatch is joined in.
  3. It backs up and removes the mismatched nucleotide.
  4. It adds the correct one.
  5. Then it carries on: about 1 mistake in 10 million survives.
The knobs and notches stand for the atoms that form hydrogen bonds (A1.2). A knob fits a notch, and a blank fits a blank.
CHGOHAHUOHGHTHAHTHCHAHOCHOUHOGHOlaggingleading5′3′oldnew?pol III5′3′primeroldnewprimasenick5′3′primerpol IprimerDNA ligaseoldnewpol IIImismatch5′3′
  1. One primer starts the leading strand.
  2. On the lagging strand: a new primer for every fragment.
  3. DNA polymerase I swaps the RNA primer for DNA.
  4. DNA ligase makes the last bond in the backbone.
  5. A mismatch at the 3′ end: what does DNA polymerase III do next?

02Dry lab: PCR and gel: whose DNA is it?

Copy a trace of DNA in a thermal cycler, run it on a gel beside a ladder, and read the bands to settle a case. Then add markers and watch the chance of a false match fall.

Your questionDo the profiles show who the father is, or whose blood was left at the scene, and how many markers do you need before you can be confident?
What you can changeThe case; how much DNA you start with; the number of PCR cycles; the three temperatures; the polymerase; the primers; the gel; how long the gel runs; how many markers you run.
What to recordFor each person and each marker: the sizes of the bands, read against the ladder, and the alleles they stand for. Then your verdict, and the chance that an unrelated person would match by luck.
Who's the father?A made-up case.A mother has asked whether eitherof two men is her child's father.The mother, the child and both meneach gave a cheek swab.Who could be the father?mothercheek swabchildcheek swabman Acheek swabman Bcheek swab25.0°Ccycle 0 of 30thermal cycler20406080100temperature / °Ccycle 1cycles 2 to 30denatureannealextend12345100 V0:00power pack−+1 ladder · 2 mother · 3 child · 4 man A · 5 man B
  1. The case: who could be the father? One tube for each sample.
  2. The thermal cycler: set the cycles and the three temperatures, then run the PCR.
  3. The gel: every sample loaded into its own well beside a ladder, ready to run. The whole bench, set up.

Find out

  • Who's the father? Run three markers. Can you decide yet? If not, use Run more markers on this case until you can. How many did it take, and what was the chance of a false match then?
  • Whose blood? Decide, then check the chance that an unrelated person would match at the markers you ran. Play a few cases with different numbers of markers and watch the graph under the bench.
  • What makes PCR work? With the father case, change one PCR setting at a time and run the gel each time: 15, 20, 25 and 30 cycles; denaturation at 85 and 95 °C; annealing at 45, 56 and 68 °C; the other polymerase. Record which settings give bands.
  • Read the ladder. Switch to the size ladder. Use the standard curve: plot log size against distance, fit a line and read a band's size from it. Work out its number of repeats (the markers' sizes are under What the model is). How close is it to the allelic ladder's answer?
  • COVID test. Call each patient. What are the two control tubes for?
  • Harder cases. Tick the box and play each case again. What goes wrong, and how would a real lab spot it?

Record

  • Each person's alleles at each marker, in the profile table, read against the ladder.
  • Your verdict, and what the bands support, marker by marker.
  • How many markers you ran, and the chance of a false match for those markers.
  • For the PCR settings, a table of the setting you changed and whether bands appeared. Download CSV keeps every reading and verdict.

With the polymerase from E. coli, the copies line never rises and the gel shows only the ladder. Why?

In a COVID run, the no-DNA tube (water, no swab) shows a faint viral band. What should the lab do?

With one marker, both men could be the child's father. With six, one of them is excluded. What does this show?

03Check

A fresh paper from the exam bank on this guide's topics each time: past-paper style questions, marked as you go, and saved to your Exam practice progress when you are signed in. It opens on biologybybradford.com, so it needs the internet.

Exam practice

Exam questions on D1.1, marked

Start 15 marks of exam questionsYour exam practice

04Downloads

This file is the guide, saved to run without the internet.

Saved from biologybybradford.com/guides/ib-biology-d1-1-dna-replication, where the guide is kept up to date with the notes, the revision guide and, for the teacher, the lesson plans and slides.

05Teacher resources

The guide as two one-hour lessons, and two more at higher level. Each has a plan to print (the timings, what to project and where to stop, the activities with what they need, what students get wrong) and slides to teach it from, in the same order, with the teacher's moves in the notes.

LessonCoversFocusChaptersLesson planSlides
1Copying the code D1.1.1 to D1.1.360 min Why cells copy DNA, how the copy stays exact, and the two enzymes that do it 1, 2, 3 Lesson planPDF 696 KB SlidesPPTX 1.2 MB
2Amplify, separate, identify D1.1.4, D1.1.560 min PCR, gels and DNA profiles, and why more markers mean fewer false matches 4, 5, 6, dry lab Lesson planPDF 766 KB SlidesPPTX 992 KB
3Two directions at one fork HL D1.1.6, D1.1.760 min Why polymerase adds only at the 3′ end, and what that means for the two new strands 7, 8 Lesson planPDF 687 KB SlidesPPTX 662 KB
4The enzymes and the checks HL D1.1.8, D1.1.960 min Primase, polymerases III and I, ligase, and proofreading 9, 10 Lesson planPDF 689 KB SlidesPPTX 1.0 MB
PDFEvery lesson planAll four plans in one PDF, with this overview at the front. 1.3 MB KEYNote template, filled inThe class’s note template with every gap filled. 1.5 MB

The note template, the revision guide and the offline copy are in Downloads, for you and the class.

PRACTISE AND REVISE

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Open a set to host it as a class game, or make a copy to edit.

How this was made. The course structure, the activities and the teaching choices come from my own classroom, my presentations and my years of teaching IB and AP Biology. I use Claude, an AI, to help write and build the interactive guides, lesson plans, slides and practice questions, and to draw the diagrams and code the interactives. I direct the work and review it against the IB and AP specifications before it goes live.