DNATechnologies

Chapter 13

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

Describe the function of a thermal cycler and how PCR results are visualized

Discuss applications of PCR technology, including uses in the field of forensics

Discuss the benefits and implications of knowing the DNA sequences of humans and other organisms

Explain how DNA is sequenced using the Sanger Method and the recent improvements that have increased the efficiency of this process

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.

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.

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.

Vocabulary

• Homologous pairs – the two “matching” chromosomes that have the same genes in the same order

• DNA replication – the process by which DNA molecules are duplicated

• in vivo –an experiment conducted in a living organism or cell; literally “in living”

• Helicase – an enzyme that functions to unwind and unzip complementary DNA strands during in vivo DNA replication

• Topoisomerase – an enzyme that acts to relieve tension in DNA strands as they unwind during in vivo DNA replication

• RNA primase – an 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 – an enzyme that, during DNA replication, creates a new strand of DNA nucleotides complementary to a template strand

• RNase H – an enzyme that functions to degrade RNA primers, during in vivo replication, that are bound to DNA template strands

Vocabulary

• in vitro synthesis – any synthesis that is done wholly or partially outside of a living organism (eg, PCR); literally, “in glass”

• Probes – the labeled DNA or RNA sequences (oligonucleotides) that are used for gene identification

• DTT – the abbreviation for dithiothreitol, a reducing agent that helps stabilize the DNA polymerase in DNA synthesis, PCR, and DNA sequencing reactions

• Template – the strand of DNA from which a new complementary strand is synthesized

• dNTP – the 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 – the abbreviation for deoxyadenosine triphosphate, the cell’s source of adenine (A) for DNA molecules

• dCTP – the abbreviation for deoxycytidine triphosphate, the cell’s source of cytosine (C) for DNA molecules

• dGTP – the abbreviation for deoxyguanosine triphosphate, the cell’s source of guanine (G) for DNA molecules

• dTTP – the abbreviation for deoxythymidine triphosphate, the cell’s source of thymine (T) for DNA molecules

• Reaction buffer – a buffer in PCR that is used to maintain the pH of the synthesis reaction

13.1 Review Questions

1. How many DNA strands does an E. coli cell contain? How many chromosomes does a human body cell contain?

2. What are homologous pairs, and where do they come from?

3. Name six enzymes involved in in vivo DNA replication.

4. How is in vitro DNA synthesis in a test tube different than in vitro DNA synthesis in an automated synthesis?

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.

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.

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.

Vocabulary

• Amplification – an increase in the number of copies of a particular segment of DNA, usually as a result of PCR

• Cross-linker – an instrument that uses UV light to irreversibly bind DNA or RNA to membrane or paper

• Microarry scanner – an instrument that assesses the amount of fluorescence in a feature of a microarray

• Primer design – a process by which a primer sequence is proposed and constructed

13.2 Review Questions

1. What is it called when DNA samples are transferred to a membrane for staining or probing?

2. How are probes used in microarrays?

3. 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.

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

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.

Vocabulary

• Primer annealing – the phase in PCR during which a primer binds to a template strand

• Extension – the phase in PCR during which a complementary DNA strand is synthesized

• Optimization – the process of analyzing all the variables to find the ideal conditions for a reaction or process

13.3 Review Questions

1. Why is Taq polymerase used in PCR instead of some other DNA polymerase?

2. What are the three parts to a thermal cycling reaction, and what is the difference in temperature between them?

3. What is it called when a PCR technician determines the best conditions for running a PCR protocol?

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

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.

Vocabulary

• Karyotyping – the process of comparing an individual’s karyotype with a normal, standard one to check for abnormalities

• VNTRs – the 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

13.4 Review Questions

1. Restriction fragment length polymorphism technology was formerly used for DNA fingerprinting. What technology is currently used for DNA fingerprinting?

2. For a DNA fingerprint, many PCR targets are used. Each target is its own VNTR. What is a VNTR?

3. 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?

13.5 DNA Sequencing

DNA sequencing, polymerase chain reaction (PCR), microarray, and bioinformatics have provided so much data that researchers must design, conduct, and report the results of their experiments in ways that are different from those that were standard just a generation ago.

DNA Sequencing

DNA sequencing includes all the techniques used to determine the order of nucleotides (A, C, G, and T) in a DNA fragment.

Vocabulary

• DNA sequencing – pertaining to all the techniques that lead to determining the order of nucleotides (A, C, G, and T) in a DNA fragment

• Dideoxynucleotide sequencing – a sequencing method that uses ddNTPs and dNTPs in a predictable way to produce synthesis fragments of varying length; also called the Sanger Method

• Dideoxynucleotides – the nucleotides that have an oxygen removed from carbon number 3, abbreviated ddNTPs

• BLAST – an acronym for Basic Local Alignment Search Tool, a program that allows researchers to compare biological sequences

• Cycle sequencing – a technique developed in the late 1990s that allowed researchers to run synthesis reactions over and over on samples, increasing the amount of sequencing product and the speed of getting results

• Human Genome Project – a collaborative 10-year project completed in 2000, which aimed to sequence the entire DNA code for the human organism

13.5 Review Questions

1. How is ddNTP different from a “regular” dNTP?

2. When preparing sequencing-reaction tubes, each of the four dNTPs are added, but just one kind of ddNTP. Which are used in the highest concentrations, the dNTPs or the ddNTPs, and why?

3. Where on the Internet may one go to compare DNA sequence data?

4. What additional instrument is required for cycle sequencing?

Questions and Comments?