(B) Amplification and detection of DNA sequences. (5) Human Genomics DNA (A) Sequencing DNA (B) Amplification and detection of DNA sequences.

Human Genomics (B) State the function of PCR Describe the steps involved in the PCR process State the role of a PCR primer State what is DNA probe is State the function of a DNA probe State how DNA probes can be detected Give examples of applications of PCR

Amplification and detection of DNA sequences Carried out by PCR Detection Arrays of DNA probes Fluorescent labelling

What is PCR? PCR stands for Polymerase Chain Reaction It is a technique for the “amplification” of DNA in vitro. This means to make multiple copies of DNA

What is PCR? PCR was developed by Kary Mullis in the mid-1980s. PCR revolutionised molecular biology, and Mullis received the Nobel Prize for chemistry for its conception in 1993. The technique enables specific sections of DNA to be amplified (replicated) in vitro, producing millions of copies from a DNA template. Mullis developed the technique manually, and it can still be carried out using water baths. However, the technique is now fully automated in laboratories, using thermal cyclers no bigger than a bread machine. The technique manipulates the cell’s natural mechanism for replication by using DNA polymerase.

How is PCR carried out? Continuous cycles of: Heating Cooling Tends to be between 30-40 cycles! Continuous cycles of: DNA is first heated to over 90°C Heating Cooling Annealing Extending ~1 min ~2 mins Specific DNA sequence extends at above 70°C DNA is then cooled to 40-60°C animation 1 animation 2 ~45 sec click through animation

Polymerase chain reaction

TASKS – 15 minutes Sort the 5 diagrams into the correct order Get your cards checked! Stick in your diagram under the title “One PCR Cycle” and label it using your diagrams! Label each step with one of the following terms: Annealing / Cooling / Extending / Melting The 15 cards describe the process of PCR. Put them into the correct order! HINT: “amplification” Copy down the paragraph under the title “Polymerase Chain Reaction” Be able to tell your partner what is happening in each picture 1 – melting 2 – cooling 3 – annealing 4 - extending

Polymerase chain reaction Amplification is the term used to describe multiple copies of DNA being produced using PCR. The first stage is melting. Melting is the term used to describe heating the DNA to over 90°C. This is to separate the 2 strands. The DNA is then cooled. This is to allow complementary primers to bind to target DNA sequences. Annealing is the term used to describe the primers binding to the 2 ends of the region to be amplified. Heat-tolerant DNA polymerase then replicates the region of DNA. The enzyme joins nucleotides together and extends the strand. Extending is the term used to describe the complementary strands of target DNA being made. Repeated cycles of heating and cooling take place in order to amplify the region of DNA. Answer to card sort

PCR problem solving 3. A forensic scientist discovered a tiny spot of blood at a crime scene. A sample taken from this spot contained 100 molecules of DNA. The sample underwent PCR cycles for 40 minutes. Use the graph to calculate how many molecules of DNA would be present after this time. Separation of DNA strands Primers anneal to target / complimentary sequences of DNA Temperature increases to allow DNA polymerase to extend DNA strands 41°C 1. What is the maximum change in temperature during this one cycle of PCR? 2. What is happening at each stage in the graph above? 25,600

Once DNA has been amplified how is it searched? DNA probes Once DNA has been amplified how is it searched?

What characteristics will DNA probes need to have? What are DNA probes? DNA probes are short single stranded fragments of DNA that are complementary to a specific sequence in DNA. (Do not get probes mixed up with the primers used in PCR.) What characteristics will DNA probes need to have?

You just wrote these down!! DNA probes Made of DNA Short Single stranded Complementary to the sequence being searched for You just wrote these down!!

How do we find out where the probes have bound to the DNA? Before the DNA probe is introduced to the DNA it is fluorescently labelled. This involves attaching a fluorescent dye to the probe, which allows for detection. For example, ethidium bromide fluoresces orange when bound to DNA and exposed to UV light. Any ideas?

Fluorescent labelling of DNA probes allows for detection

Medical and Forensic Applications TASK: List as many fields as you can in which DNA probes along with PCR can be used! Each group should a box from the next slide and DISCUSS how DNA probes along with PCR would be used in the example

DNA profiling: A woman is found dead in a hotel room DNA profiling: A woman is found dead in a hotel room. Scene of crime officers find some skin cells under her fingernails. A man who fits the description of someone seen leaving the room a few hours earlier is arrested, but he claims he has never met the victim. What would be the role of PCR? DNA profiling: The illegal trade of endangered species on the black market is highly profitable for those involved. How would customs officers employ PCR in helping to prevent this activity? DNA profiling: The similarities and differences in DNA can provide information on how long ago two organisms evolved into different species. A new species of plant is discovered in the Amazon rainforest. How would scientists use PCR to help identify which plants it is related to? Disease diagnosis: Certain genetic mutations that increase the risk of a woman developing breast cancer have been identified. If breast cancer runs in a family, a woman can be tested to see if she carries these mutations. How would PCR be beneficial? Archaeological analysis: Some dinosaur bones have been discovered. Although DNA degrades over time, some is found in the marrow of the bones. Scientist wish to find out which species of dinosaur they are from. How would PCR be beneficial? Population studies: The frequency of alleles of a particular gene can vary over time. Population genetics studies how this frequency changes. How would PCR be beneficial in this process?

Applications of PCR PCR is used widely. A few examples are listed below: DNA profiling/fingerprinting: PCR is used to rapidly identify individuals. Specific regions of DNA known to vary between individuals are amplified using fluorescently labelled primers and then analysed using capillary gel electrophoresis. Profiling used not only in forensics but also in plant variety identification, paternity testing and evolutionary biology. Disease diagnosis: DNA sequences known to indicate certain genetic disorders or diseases are amplified using PCR for the purposes of diagnosis. Archaeological analysis: Ancient DNA, degraded over the years, can be amplified and used in archaeological, paleontological and evolutionary research. Population studies: Analysis of human or other species’ population genetics can be quickly performed using PCR analysis. Sequencing: DNA sequence analysis previously took place following lengthy cloning experiments, which have now been replaced by the use of PCR.

Identifying if a particular individual left blood at a crime scene DNA profiling allows the identification of individuals through comparison of regions of the genome with highly variable numbers of repetitive sequences of DNA. So if a tiny sample of blood is left at a crime scene, the DNA it contains can be amplified using PCR. A probe can then be created, which binds to one of these highly variable sequences. If a suspect is apprehended their DNA can be tested with the same probe to look for the sequence in question.

Making a diagnosis of disease status or risk of disease onset A cell sample from a patient can be screened for the presence or absence of a particular sequence, eg a mutation in a gene. This information can be used to diagnose a condition or describe the likelihood of a condition developing. For example, women with a family history of cancer can be tested for mutations in the BRCA 1 and BRCA 2 genes. Mutations in these genes can increase the risk of developing breast or ovarian cancer.

Who is Child X’s father? Who could be child Y’s father? Man P Man Q

DNA profiling / fingerprinting Archaeological analysis Applications of PCR DNA profiling / fingerprinting Archaeological analysis Population studies Disease diagnosis Sequencing

Applications of PCR Applications of DNA profiling allow the identification of individuals through comparison of regions of the genome with highly variable numbers of repetitive sequences of DNA. This could be to identify criminals or biological parents By screening a cell sample from a patient for the presence or absence of a particular sequence, a diagnosis of disease status or risk of disease onset can be made.

Human Genomics (B) State the function of PCR Describe the steps involved in the PCR process State the role of a PCR primer State what is DNA probe is State the function of a DNA probe State how DNA probes can be detected Give examples of applications of PCR