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 DNA Template Primer
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 DNA Polymerase Reaction Buffer
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 PCR Amplification
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. Challenges in PCR Technology
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 Forensics
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 Reaction How 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. The PCR Reaction What do you need?
What is needed for PCR?
Template - the DNA to be amplified
Primers - 2 short specific pieces of DNA whose sequence flanks the 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)
The PCR Reaction What do you need?

28. What do we use? Reagents and supplies Equipment 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