MOLECULAR BIOLOGY OVERVIEW NUCLEIC ACIDS: THE BASICS

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1 MOLECULAR BIOLOGY OVERVIEW NUCLEIC ACIDS: THE BASICS Richard L. Hodinka, Ph.D. University of South Carolina School of Medicine Greenville Greenville Health System, Greenville, SC

2 Objectives of Talk Describe the structure of DNA and RNA molecules Apply basic genetic concepts and terms to molecular diagnostic technologies Discuss the various parameters needed to successfully perform PCR

3 Molecular Biology Molecular basis of biological activity Simply put, the study of nucleic acids In the clinical laboratory, molecular techniques are designed for handling of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) The ability to amplify these nucleic acids has made it possible to analyze virtually any piece of DNA in nature

4 Eukaryotic and Prokaryotic Cells Illustration from NCBI Science Primer: What is a Cell?

5 Central Dogma of Molecular Biology DNA transcription RNA translation Protein National Human Genome Research Institute (NHGRI) Genetic Illustrations

6 Let s look at DNA molecule

7 DNA DeoxyriboNucleic Acid DNA belongs to a class of molecules called nucleic acids; they are polymers of four kinds of building blocks or nucleotides Most genes are made of DNA DNA normally in form of a double-stranded helix (a twisted latter); each chain of the double helix is made of repeating nucleotides Each nucleotide is made up of three parts: Phosphate groups Pentose (5 carbon or 5-sided) sugar 4 subunits termed nitrogenous (nitrogencontaining) bases

8 Nitrogenous Bases Purines (Two Rings) Pyrimidines (One Ring)

9 Sugar Life Sciences Cyberbridge (http://cyberbridge.mcb.harvard.edu/dna_1.html)

10 Nucleosides Adenosine (A), Guanosine (G), Cytidine (C), and Thymidine (T)

11 Schematic of Nucleotide Phosphate + Sugar + Base = Nucleotide 2 -Deoxyribonucleotide Deoxyadenosine 5 -triphosphate (datp) Life Sciences Cyberbridge (http://cyberbridge.mcb.harvard.edu/dna_1.html)

12 DNA Base Nomenclature Base Nucleoside Nucleotide Acronym Base Ring Structure Adenine (A) Adenosine Adenosine Triphosphate datp Purine Guanine (G) Guanosine Guanosine Triphosphate dgtp Purine Thymine (T) Thymidine Thymidine Triphosphate dttp Pyrimidine Cytosine (C) Cytidine Cytidine Triphosphate dctp Pyrimidine

13 Phosphodiester Linkages to Build Polynucleotide Phosphodiester bonds between the 3 C of sugar and 5 C of next sugar

14 Base Pairing National Human Genome Research Institute (NHGRI) Genetic Illustrations

15 DNA Replication Each strand of DNA acts as template for production of new strand Replication machinery includes large number of different proteins present in cell Use dntps as precursors Double stranded DNA unzips and new double helix is formed from each template parent strand DNA polymerase binds the correct nucleotide into place by base pairing Process is semi-conservative

16 RNA RiboNucleic Acid Almost always found as a single strand molecule, and not double stranded Looks like DNA except it contains ribose rather than deoxyribose sugar and uracil instead of thymine Main types of RNA in cells: Messenger RNA (mrna) carries the genetic information out of nucleus; converted into protein Ribosomal RNA (rrna) along with a series of proteins, makes up the ribosomes of a cell Transfer RNA (trna) for protein synthesis

17 RNA Structure

18 Summary: DNA Versus RNA DNA RNA Sugar Deoxyribose Ribose Bases Adenine (A) Adenine (A) Cytosine (C) Cytosine (C) Thymine (T) Uracil (U) Guanine (G) Guanine (G) No. of Strands Double Usually single Heat Stable Yes No

19 Nucleic Acid Amplification If DNA or RNA sequence of pathogen is known, short portion of sequence unique to pathogen can be identified This is the TARGET or TEMPLATE SEQUENCE With this knowledge, small pieces of nucleic acid (OLIGONUCLEOTIDES) can be used to synthesize and detect new DNA or RNA

20 Target DNA or RNA Highly conserved region of the genome that you want to detect In our laboratory, targets range in size from as small as 55 bp (RSV A) to as large as 207 bp (Rhinovirus)

21 Typical Polymerase Chain Reaction (PCR) PCR happens in cycles with three basic steps: PCR allows a small amount of the DNA molecule to be amplified exponentially. 40 cycles = 1x10 12 copies of target! (theoretically)

22 Typical Polymerase Chain Reaction (PCR)

23

24 Molecular Amplification

25 Complementary DNA (cdna) When using PCR to amplify RNA (e.g., from RNA-containing viruses or during expression of a gene as RNA), the RNA must first be turned into DNA Reverse transcriptase (RT) enzyme is used to make a DNA copy of the RNA The DNA is complementary to the RNA and is called cdna cdna is then used in PCR

26 Nucleic Acid Template A master copy of DNA or RNA to direct the synthesis of the new strand of nucleic acid; also called a TARGET sequence Template (Target) can be DNA or RNA Important to select conserved region of genome Want target to be genetically stable Bacteria Circular ds chromosomal DNA Many have circular plasmid DNA (extrachromosomal) Ribosomal mrna Viruses DNA or RNA but not both DNA can be ss or ds, linear or circular RNA can be ss or ds, (+) sense (like mrna) or (-) sense, segmented or linear

27 Standard PCR Master Mix Target Nucleic Acid Set of Primers Probe (if used for detection) dntps Enzymes Taq DNA polymerase Reverse transcriptase (for RNA) Reaction Buffer 10 mm Tris-HCl (ph 8.3) MgCl 2 50 mm KCl 0.001% gelatin RNase Inhibitor

28 Oligonucleotides Synthetic short segments of nucleic acids Are specific (complementary to target sequence) Two types are PRIMERS and PROBES

29 Primers and Probes Primers and probes are of defined length and are usually DNA Primers anneal (bind) to target sequence during amplification; DNA polymerase will not work without small section of doublestranded DNA to initiate or prime new DNA synthesis Probes hybridize (bind) to target sequences during detection of amplified product (target)

30 Primers to Amplify Nucleic Acid Short segments of DNA oligonucleotides that anneal to the target during replication Starting point for DNA synthesis - this is where polymerase binds

31 Degenerate Primers Set of primers which have number of options at several positions in the sequence to allow annealing to and amplification of variety of related sequences. An example: TCCAAGTYAACVCCT Y = C or T V = A or C or G; not T

32 Probes to Detect NA Amplification Probes are highly specific to the target being amplified During target amplification, added probe will bind to middle of target between the flanking primers Probes normally contain fluorescence molecules to detect the amplified target

33 Enzymes Proteins that catalyze a biochemical reaction DNA polymerase and reverse transcriptase are some of the most commonly used enzymes in molecular biology, particularly in PCR Nucleases (DNase or RNase) are found in biological fluids and digest nucleic acids; during purification of nucleic acids, must quickly inactivate these before they chew up the purified DNA or RNA Uracil-N-glycosylase (UNG) - AmpErase Remember, enzymes always end with the suffix ase

34 DNA Polymerase The Great Replicator Synthesizes complementary DNA by linking datp, dctp, dgtp, and dttp in specific order using singlestranded DNA as template Done in 5 to 3 direction beginning with primer with free 3 -OH Illustration from Chapter 7, Recombinant DNA and Biotechnology, H. Kreuzer and A. Massey

35 Reverse Transcriptase RNA-dependent DNA polymerase Synthesizes DNA complementary to an RNA template Done in 5 to 3 direction starting with primer with 3 -OH Requires dntps Makes PCR analysis of RNA possible

36 Questions Anyone?

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