DNA Replication & PCR Notes PDF

ANSWERS TO PUPIL NOTES Unit 1: DNA and the Genome Key Area 1.2 The Replication of DNA (a) Replication of DNA 1 Explain the importance of DNA replication prior to cell division. 2 State 5 things that must be present for DNA replication. 3...

ANSWERS TO PUPIL NOTES Unit 1: DNA and the Genome Key Area 1.2 The Replication of DNA (a) Replication of DNA 1 Explain the importance of DNA replication prior to cell division. 2 State 5 things that must be present for DNA replication. 3 Describe the stages of DNA replication. 4 Describe what a primer is and explain its role in DNA replication. 5 Name 2 enzymes in involved in DNA replication. 6 Explain the role of each enzyme in DNA replication. 7 Describe the direction of replication on each DNA strand. 8 Explain why the direction of DNA replication is always in this direction. (b) Polymerase Chain Reaction (PCR) 1 Describe the purpose of PCR. 2 Explain how primers are chosen for a particular PCR. 3 Explain what is involved in the ‘thermal cycling’ of PCR. 4 Describe the role of heat tolerant DNA polymerase in PCR. 5 Describe 3 practical applications of PCR. 1  Glossary Replication Ligase Template strand DNA polymerase Heat-tolerant DNA polymerase Primer PCR Leading strand Lagging strand Word Definition Template strand DNA strand on which a complementary copy is made Lagging strand DNA strand that is replicate in fragments Leading strand DNA strand that is replicated continuously Heat-tolerant DNA polymerase The enzyme used in PCR that doesn’t denature at high temperatures. DNA polymerase The enzyme that adds free complementary DNA nucleotides during replication of DNA Ligase Enzyme that joins DNA fragments to make the lagging strand Replication Formation of identical copies of DNA molecules PCR Repeated cycles of heating and cooling to amplify the target region of DNA. Primer Short complementary strand of DNA needed to initiate the process of DNA replication. 2  Key Area 1.2a DNA Replication DNA is a unique molecule because it is able to direct its own replication and produce an exact copy of itself. This is essential prior to cell division so that each daughter cell contains the exact same genetic information as the original parent cell. Basic Summary of DNA Replication 1. Parent DNA strand unwinds and hydrogen bonds between the bases break. 2. Each parent strand acts as a template and individual nucleotides (present in the nucleus) align with the nucleotides on the template according to the base-paring rule. New hydrogen bonds form. 3. The sugar-phosphate backbone forms on the new strand. 4. Each new DNA molecule is identical to the original parent. Requirements for DNA Replication For DNA replication to take place, the nucleus must contain: 1. DNA template 2. Primers 3. Free DNA nucleotides 4. Enzymes 5. ATP 3 Stage 1 DNA unwinds and the weak hydrogen bonds between the base-pairs break to form two template strands. A primer is a short strand of nucleotides needed to start DNA replication. A primer binds to the 3’ end of the template DNA Therefore, the process of DNA replication is slightly different on each strand. The strand with the free 3’ end is called the ‘leading strand’. The strand with the free 5’ end is called the ‘lagging strand’. leading strand lagging strand 3’ end primer 5’ end 4  Stage 2 – Leading Strand  DNA is replicated by the enzyme DNA polymerase  For the enzyme to work it must add new nucleotides to a pre-existing chain and this is why the primer is important.  DNA polymerase can only add nucleotides in one direction (from 3’ end) of a DNA strand resulting in the leading strand being replicated continuously. leading strand primer Stage 2 – Lagging Strand Since DNA polymerase is only able to add nucleotides to the free 3’ end of a growing strand, the template strand that has the 5’ end has to be replicated in fragments (discontinuously) each starting at the 3’ end of a primer. Then the fragments are joined together with the help of an enzyme called ligase. primer 5  Key Area 1.2b Polymerase chain Reaction (PCR) The Purpose of PCR The Polymerase Chain Reaction (PCR) is a technique for the amplification of DNA in vitro. This technique allows scientists to easily and cheaply turn a single strand of DNA into millions of copies which can then be used for analysis. Requirements for PCR 1. Sequence of DNA (to act as a template 2. Primers (required to start replication) 3. DNA nucleotides 4. Heat tolerant DNA polymerase (an enzyme that will not denature at high temperatures) Primers in PCR During PCR primers are used. The primer used is selected to be complementary to a specific target sequence at the 3’ end of the DNA to be replicated. This allows PCR to be highly specific to a particular sequence of DNA. Two primers are needed as each primer targets a different strand 6 PCR Cycle 1: The process of PCR can be described as ‘thermal cycling’ as the DNA is repeatedly heated and cooled. Step 1: Step 1: The DNA is heated to between 92 -98⁰C to break the hydrogen bonds between base pairs. This allows for the separation of the DNA strands. Step 2: Step 2: The DNA is then cooled to 50- 65⁰C to allow the primers to bind to their specific target sequences at the 3’ ends. 7 Step 3: Step 3: The DNA is then heated to 70⁰C - 80 for heat-tolerant DNA polymerase to replicate the region of DNA The product is two identical copies of DNA at the end of the first cycle. PCR – Cycle 2 and onward cycles The cycle is repeated using the two strands produced in cycle one. The product of cycle two is therefore four identical copies of DNA. This process is then repeated many times (around 20-30 times) with each cycle doubling the quantity of DNA present at the start of that cycle. 8 Practical Applications of PCR PCR has revolutionised research in many areas of science. It has been used to amplify DNA from many sources such as:  Amplification of tiny quantities of DNA from blood, semen or tissue from a crime scene for DNA fingerprinting  Embryonic cells for prenatal diagnosis of genetic disorders  Paternity testing 9  1.2 Past Paper Questions 1 Which of the following molecules are required in the replication of the lagging strand of a DNA molecule? A DNA polymerase & ligase only B DNA polymerase & primers only C Ligase and primers only D DNA polymerase, ligase & primers 2 Each cycle of PCR takes 5 minutes. If there are 1000 DNA fragments at the start of the reaction, how long will it take for the number of fragments produced by the reaction to be greater than 1 million? A 15 minutes B 35 minutes C 50 minutes D 55 minutes During DNA replication two new daughter strands are synthesised using the original strands 3 as templates. a) State why the antiparallel nature of the DNA molecules results in one of the strands being synthesised in short fragments. (1) Nucleotides added to 3’ end OR polymerase (only adds to 3’ end OR Polymerase works from 5’ to 3’ OR DNA/it is replicated from 5’ to 3’ b) Template DNA, enzymes and ATP are necessary for DNA replication. State one other substance required. (1) (DNA) nucleotides OR primers 10  c) Explain why cells need to carry out DNA replication. (1) 4 Two heat-tolerant DNA polymerases used in polymerase chain reactions (PCR) are Taq & Pfu. Pfu has ‘proof reading’ activity. It checks that the correct nucleotides are inserted during replication of a target sequence and then corrects any errors. The graph shows the temperatures during a single PCR cycle required to amplify a target sequence using Taq & Pfu. 11  (a) (i) Calculate the time taken for 16 copies of the target sequence to be made from one DNA fragment using Taq polymerase. (1) Space for calculation WORKING: 4 cycles needed to make 16 copies. 12 minutes 1 cycle takes 3 mins 4x3 = 12 (a) Continued (ii) Identify the time period during which primers bind to the original DNA fragment. (1) From 1.1 to 2 minutes (b) A scientist was planning to amplify DNA using PCR. State which DNA polymerase should be used and describe the advantage of using this polymerase. (1) DNA polymerase: Taq OR Pfu Advantage: Advantage: Takes less time to amplify Or replicated faster OR cheaper has less heat is required OR taq takes 3 minutes & Pfu takes 4 minute OR Pfu corrects errors (c) Explain the importance of using heat-tolerant DNA polymerases in PCR. (1) So it does not denature (NOT: so it can work at high temperatures) 12 13