Making DNA Molecules

1. Making DNA Molecules Chapter 13

2. Learning Outcomes • Describe the process of semiconservative DNA replication in cells and compare and contrast this method with DNA synthesis in the laboratory • Discuss the uses of synthesized oligonucleotides and identify the attributes of good primers • Explain the steps of PCT and discuss the components and optimization of the process • Discuss the function of a thermal cycler and how PCR results are visualized • Describe applications of PCR technology, including uses in the field of forensics

3. 13.1 Making DNA Molecules – DNA Synthesis • A DNA molecule, at any given moment, could be involved in: • DNA replication • Transcription DNA and Chromosomes DNA molecules directly code for all the RNA and protein molecules that a cell synthesizes. The 44 chromosomes in human cells are actually 22 homologous pairs, plus 2 sex chromosomes.

4. DNA Replication A human body is estimated to have over 20 trillion cells. These cells all originate from a single fertilized egg cell by means of DNA replication. DNA Template A template DNA is the strand from which a new strand is synthesized. Primer A primer is a short piece of DNA or RNA that is complementary to a section of template strand.

5. Nucleotides Nucleotide triphosphates are the reactants used as the sources of A, C, G, and T for the new strand. DNA Polymerase Polymerase builds large molecules (polymers) from smaller molecules (monomers). Reaction Buffer Reaction buffer is used to maintain the pH of the synthesis reaction.

6. Vocabulary • Homologous pairs – two “matching” chromosomes that have the same genes in the same order • DNA replication – process by which DNA molecules are duplicated • in vivo– referring to an experiment conducted in a living organism or cell; literally “in living” • Helicase – enzyme that functions to unwind and unzip complementary DNA strands during in vivo DNA replication • Topoisomerase – enzyme that acts to relieve tension in DNA strands as they unwind during in vivo DNA replication • RNA primase – enzyme that adds primers to template strands during in vivo DNA replication • Primer – a short piece of DNA or RNA (15 – 35 bases) that is complementary to a section of template strand and acts as an attachment and starting point for the synthesis strand during DNA replication • DNA polymerase – enzyme that, during DNA replication, creates a new strand of DNA nucleotides complementary to a template strand • RHase H – enzyme that functions to degrade RNA primers, during in vivo replication, that are bound to DNA template strands

7. Vocabulary • in vitro synthesis – any synthesis that is done wholly or partially outside of a living organism (eg, PCR); literally, “in glass” • Probes – fluorescently labeled DNA or RNA sequences (oligonucleotides) that are used for gene identification • DTT – abbreviation for dithiothreitol, a reducing agent that helps stabilize the DNA polymerase in DNA synthesis, PCR, and DNA sequencing reactions • Template – strand of DNA from which a new complementary strand is synthesized • dNTP – abbreviation for nucleotide triphosphates, which are the reactants used as the sources of A, C, G, and Ts for a new strand of DNA • dATP – abbreviation for deoxyadenosine triphosphate, the cell’s source of adenine (A) for DNA molecules • dCTP – abbreviation for deoxycytidine triphosphate, the cell’s source of cytosine (C) for DNA molecules • dGTP – abbreviation for deoxyguanosine triphosphate, the cell’s source of guanine (G) for DNA molecules • dTTP – abbreviation for deoxythymidine triphosphate, the cell’s source of thymine (T) for DNA molecules • Reaction buffer – buffer in PCR that is used to maintain the pH of the synthesis reaction

8. 13.1 Review Questions • How many chromosomes does an E. coli cell contain? How many chromosomes does a human body cell contain? • What are homologous pairs, and where do they come from? • Name six enzymes involved in in vivo DNA replication. • How is in vitro DNA synthesis in a test tube different than in vitro DNA synthesis in an automated synthesis?

9. 13.2 DNA Synthesis Products • DNA is commonly synthesized for these applications • Probes • Primers • PCR amplification Probes Probes are relatively short pieces of DNA (or RNA) with a nucleotide sequence complementary to another sequence being searched for.

10. Blotting Samples are transferred from the gel to a membrane or specially treated paper. Microarrays Microarrays are assemblies of large numbers of samples of DNA, or even RNA samples.

11. Constructing Primers Primers are constructed to recognized a particular section of DNA. This is called primer design. PCR Amplification Primers are used when trying to mark, identify, or amplify a piece of DNA.

12. Vocabulary • Amplification – increase in the number of copies of a particular segment of DNA, usually as a result of PCR • Cross-linker – instrument that uses UV light to irreversibly bind DNA or RNA to membrane or paper • Microarry scanner – instrument that assesses the amount of fluorescence in a feature of a microarray • Primer design – process by which a primer sequence is proposed and constructed

13. 13.2 Review Questions • What is it called when DNA samples are transferred to a membrane for staining or probing? • How are probes used in microarrays? • Design a primer that would be good for recognizing the beginning of the following “sequence of interest.” Describe why your primer is a good one. • 3’ACACAGGATACGTGCTGCTCAATGCCATGATAGCCGGTCACAAGC- • TAATCCGATTTCGCGCAAATTCCTAAATTCGCTAAAGC- • GAATCTTCAGGAAGGAACCCCGAAGGCCTTTT-5’, and so on.

14. 13.3 Polymerase Chain Reaction Polymerase chain reaction (PCR) is a method by which millions of copies of a DNA segment can be synthesized in a test tube in just a few hours. Performing a PCR Reaction • Reaction buffer: Maintains pH • Forward primers: Recognize one end of the fragment to be amplified • Reverse primers: Recognize the other end of the fragment to be amplified • Taq polymerase: Special DNA polymerase that remains active at very high temperatures • dNTPs: The four deoxynucleotides (A, C, G, T) • Magnesium chloride (MgCl2): Necessary cofactor for polymerase activity

15. Cyling Program The cycling program chosen depends on the type of sample to be amplified. Challenges in PCR Technology • DNA samples are often compromised. • Concentration of reagent, and the time and temperatures of the thermal cycling program may affect the results.

16. Vocabulary • Primer annealing – phase in PCR during which a primer binds to a template strand • Extension – phase in PCR during which a complementary DNA strand is synthesized • Optimization – process of analyzing all the variables to find the ideal conditions for a reaction or process

17. 13.3 Review Questions • Why is Taq polymerase used in PCR instead of some other DNA polymerase? • What are the three parts to a thermal cycling reaction, and what is the difference in temperature between them? • What is it called when a PCR technician determines the best conditions for running a PCR protocol?

18. 13.4 Applications of PCR Technology • Forensics/criminology • Missing children/soldiers • Paternity/maternity cases • Medical diagnostics • Therapeutic drug design • Phylogeny/evolutionary studies • Animal poaching/endangered species

19. DNA Fingerprinting PCR technology came into public spotlight during the 1992 O.J. Simpson murder trial. Forensics Forensics is the application of biology, chemistry, physics, mathematics, and sociology to solve legal problems.

20. Vocabulary • Karyotyping – process of comparing an individual’s karyotype with a normal, standard one to check for abnormalities • VNTRs – abbreviation for variable number of tandem repeats, sections of repeated DNA sequences found at specific locations on certain chromosomes; the number of repeats in a particular VNTR can vary from person to person; used for DNA fingerprinting • Forensics – application of biology, chemistry, physics, mathematics, and sociology to solve legal problems including crime scene analysis, child support cases, and paternity

21. 13.4 Review Questions • Restriction fragment length polymorphism technology was formerly used for DNA fingerprinting. What technology is currently used for DNA fingerprinting? • For a DNA fingerprint, many PCR targets are used. Each target is its own VNTR. What is a VNTR? • Why would looking for the persons responsible for sneaking endangered species (rare birds, for example) into the United States be considered a job for a forensic scientist?

22. Questions and Comments?

23. PCR(Polymerase Chain Reaction)

24. PCR – Polymerase Chain Reaction has many applications • PCR is commonly used to produce many copies of a selected gene segment or locus of DNA. • In criminal forensics, PCR is used to amplify DNA evidence from small samples that may have been left at a crime scene. • PCR can be used to amplify DNA for genetic disease screening

25. The PCR ReactionHow does it work? Heat (94oC) to denature DNA strands Cool (56oC) to anneal primers to template Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA Repeat 40 cycles

26. PCR • 94 C: Denature DNA • 56 C: Anneal Primers to Template • 72 C: Activates Taq Polymerase • Repeats 31 times

27. What is needed for PCR? The PCR ReactionWhat do you need? • Template - the DNA to be amplified • Primers - 2 short specific pieces of DNA whose sequence flanksthe target sequence • Forward • Reverse • Nucleotides - dATP, dCTP, dGTP, dTTP • Magnesium chloride - enzyme cofactor • Buffer - maintains pH & contains salt • Taq DNA polymerase – thermophillic enzyme from hot springs (Thermus aquaticus)

28. What do we use? • Reagents and suppliesEquipment • and supplies • Genomic DNA sample (5 µL) P-20 pipette and tips • Master mix I (10 µL/reaction) Thermal cycler • 2.5 µL 10x PCR buffer w/o MgCl2 • 0.5 µL dNTP’s (10 mM) • 2.5 µL Forward primer (4pM/ µL) • 2.5 µL Reverse primer (4pM/ µL) • 0.15 µL Taq polymerase • 1.85 µL ddH2O • Master mix II (10 µL/reaction) • 0.75 µL MgCl2 (50 mM) • 9.25 µL ddH2O

29. Expected Results of PCR--Example • Homozygous Alu + • Homozygous Alu – • Heterozygous • Marker

30. Expected Results