BIOL3046 Part 1 - Nucleic Acids & Their Detection (PDF)

BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE BIOL3046 Foundation of Bioanalysis PART I – Nucleic acids and their detection...

BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE BIOL3046 Foundation of Bioanalysis PART I – Nucleic acids and their detection Dr. Patrick YUE Email: [email protected] Phone: 34115832 Office: RRS833 _____________________________________________________________________ Learning Objectives: - To review the structures, types and functions of nucleic acids. - To describe the characteristics of DNA and RNA. - To describe the difference between DNA and RNA. - To recognize the key features of nucleic acids required for bioanalysis. - To learn the basic principles of nucleic acid amplification using polymerase chain reaction (PCR). - To learn the requirements and steps involved in PCR. - To review the basic concept of generically modification, recombinant DNA technology and genetically modified organisms (GMO). - To learn how to test GMF using PCR - To review the concept of gene expressions and its regulatory mechanisms. - To learn how to detect specific gene expressions using Reverse transcription- polymerase chain reaction (RT-PCR). - To learn the basic principles of real-time quantitative reverse transcription PCR (qPCR) - To learn the working mechanism of common qPCR platform. _____________________________________________________________________ DNA chromosome DNA vs Gene vs - double helix - consist of DNA Gene -H-bond - ATCG ↓ called e - polymer structure of DNA with function form giant - a fragment - visible DNA Gene code for protein , regulatory function - observed it under light microscope ⑮ find DNA ? Where can we A blood (contain nucleic acid) 1 - nucleus , 4) biomolecule  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE Nucleic acids - are biological molecules essential for life - function in encoding, transmitting and expressing genetic information - include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). * Forms appearance affect biochemical may activities and functions. * sequence * formalogy L protein synthesis - translation transcription , Deoxyribonucleic acids (DNA) molecule - long > - a long polymer made from repeating units called nucleotides - a nucleotide is composed of a nucleobase (nitrogenous base), a five-carbon sugar (2'-deoxyribose), and phosphate groups - the phosphate group on each nucleotide are covalently attached to 5’- hydroxylic group of sugar (phosphodiester bond) ; and form a polynucleotide chain - the phosphate residue at the 5’ position of one sugar bonds to the 3’-hydroxyl group of another sugar - a strand is formed with sugar & phosphate units as a backbone and the nucleobases as side groups - to give a sequence a start and an end point, it is read from the nucleotide with free P-group (5’-end) to the nucleotide with free 3’-hydroxyl group (3’-end) 2  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE - overall charge : O nucleoside - net charge to : ) high temp of phosphate break -96 % OH link capply energy ~ opposite nucleotide -12345 5'Carbon · hydrogen bond = phosphodiet bond 3 #strand j F double strand forms helix  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE - there are two kinds of nitrogen bases in DNA: - nine-membered double-ringed purines - adenine (A) and guanine (G) - six-membered single-ringed pyrimidines - cytosine (C), thymine (T), uracil (U) - two nucleotides on opposite complementary DNA strands are connected via hydrogen bonds. H-bond 2-H-bond spell ! 3-H-bond - Naming of DNA strand Memorise (transcription) converting to into mRNA * DNA transcript (messager RNA) mRNA > - protein (translation) 4  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE Forms of DNA - most of the biologically active DNA exists in Watson-Crick helical form (B-form DNA) - double-helix is able to assume other forms depending upon varying environmental conditions - 6 forms - A,B,C,D,E,Z 5  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE Reading a gene sequence Gene control. protein synthesis T - U amino acid 3 matching (basic unit of protein) - ATCG - DNA 1 creplication) - TAG)- - A v CG - mRNA 6  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE - e.g. Proto-oncogene Kras - T = E S # & wi & - E S # - - - ↳ ↑ # > - ~ E I = e - = IE e = = - ⑤ T v E & ↳ ~ N j I = F - E ↳ X ↓ I - W > - - = - - · · E ↑ T ↳ & - ↑ ~ i = E - # · F · 1 5 & -- - i ~ 8 5v = 7  BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE Classwork 8  technique BIOL3046 PART I – Biomolecules and their detection Dr. P YUE : pCR * amplify gene Packaging of DNA - Visible form of DNA - chromosome ① diagnosis minor groove major groove ↓ ↓ 9  BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE Ribonucleic acids (RNA) - with similar structure to DNA - there are at least three classes of RNA molecules: ribosomal RNA (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA) + miRNA DN/ remove oxygen - the nitrogenous base thymine (T) is replaced with uracil (U) in RNA methyl group AV Gl - polynucleotide chain - base pairing - single or double strand - linear / 3-D conformation 10  BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE Types and functions of RNAs - message RNA (mRNA) – carries information about a protein sequence to the ribosomes - it is coded so that every 3 nucleotides (codon) correspond to one amino acid - ribosomal RNA (rRNA) – catalytic component of ribosomes - rRNA and protein combine to form ribosome - ribosome binds mRNA and carries out protein synthesis - transfer RNA (tRNA) – small RNA chain ~80 nucleotides, with an anticodon translated region - it transfers a specific amino acid to a growing polypeptide chain at the D , anti-it IIII AAA ribosomal site of protein synthesis during translation - codon on the mRNA is recognized by the anticodon of the tRNA codon m & ↓ frame ↑ - microRNA (miRNA) – ~22 nucleotides long RNA 5' untranslated 3 - regulate the expression of genes by binding to the 3'-untranslated regions regions (3'-UTR) of specific mRNAs - a novel mechanism of posttranscriptional gene regulation in in tRNA tRNA 11  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE stop code start code match with tRNA ↑ untranslated region wish e ⑰ tRNA - mRNI mRNA rRNA 12  BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE miRNA very short RNA > - 1 miRNA > - targe many genes I miRNA can regulate many gene expressions 13  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE - Protein synthesis - translation - mRNA, tRNA, rRNA work together speed up and process increase working efficiency - Protein synthesis, maturation and delivery -Protein synthesis Protein modification ① ② * tein packaging/ delivery MINA- exosomes shortcting 14 miRNA very 22 nucleotideantch tos'-VTR  into chop  crotein disease amino ↓ > - function > - > structure affect acid - BIOL3046 PART I – Biomolecules and their detection Dr. P YUE X changed - Codon directory gene ↓ codon - e.g. ↓ anticodon ↓ polypeptide Alanine Threonine Glutamic Leucine Arginine Serine chair ↓ A TELRS protein group ↓ 1 I code ↓ 15  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE DNA vs. RNA - Cytosine (C) occasionally converts into uracil (U) by deamination  it happens around 100 times per cell, per day. - If this “U” is not removed, at the next replication it will act as a template for are DNA an adenine (A) on the new strand, and there will have been a mutation from G very stableaeem1 to A ic - Having thymine (T) as the regular base in DNA makes it e asy for a cell to spot areinstable a deamination, because U should not be there at all. The cell then removes RNA e. the U with a DNA repair enzyme (e.g. uracil glycosylase). very - If we use uracil in DNA it would not be easy to decide how to repair that error. deamination thymine is a of L methylated analogue - Luracil O O - Thymine is a methylated analogue of uracil, DNA can be protected from nuclei by methylation (thymine being " better protected“), and therefore more suited to the storage role of DNA. - This methylation ensures DNA unrecognizable to many nucleases, thus it could not be attacked easily. - Methylating the nucleotides before they are incorporated ensures that entire strand of DNA is protected. - Uracil exists as both uridine (U) and deoxy-uridinine (dU), thymind only exits as deoxy-thymidine (dT); uracil can base-pair with almost any other base, but methyl-group restrict thymine to pairing only with adenine, this increase the DNA replication efficiency by reducing mismatching and mutation. - Thymine has greater resistance to photochemical mutation, making the genetic message more stable. Dimerization of thymine can be partially reversed, recovery of uracil dimerization is less effective. 16  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE - Sample collection and storage 17  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE DNA amplification - Polymerase chain reaction (PCR) - can amplify a desired DNA sequence of any origin (virus, bacteria, plant, or human) hundreds of millions of times in a matter of hours - is highly specific, easily automated, and capable of amplifying minute amounts of sample - has major impact on clinical medicine, genetic disease diagnostics, forensic science, and evolutionary biology. - e.g. BRCA1 gene test can't suppress 18  BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE PCR – Principle and requirements - Principle - double stranded DNA of interest is denatured to separate into 2 individual strands - each strand act as template and allows to hybridize with a primer (specific short fragment of single stranded DNA) - the polymerase starts synthesizing new strands of DNA - these three steps are repeated - Requirements - target DNA (100 – 35,000 bp in length) - primers (synthetic oligonucleotides, 17 – 30 nucleotides in length) - four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP) - thermo stable DNA polymerase limited copy very undetectable leve I unlimited copy detectable level PCR – 6 major steps - Initialization - heating reaction to temp. ~94 – 96oC for ~1 min - Denaturation - the temp. is raised at ~96oC for ~30 sec. to melt double-stranded DNA by breaking H-bond among complementary bases -Annealing Xencyme - decrease temp. to ~50-65o C for 20-40 sec., primers bind with target DNA template - annealing temp. is ~ 3-5o C below the Tm of the primers used - Synthesis / extension - Taq polymerase has optimal activity temp. at ~75-80oC - DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the 19  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. - Final elongation - at ~ 70-75oC , 5 -15min, to ensure any remaining single-stranded DNA is fully extended - Final hold - temp. keep at 4oC until collection I template a strand a - enzyme commandate temp. high 20  BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE - Cyclic reactions and DNA amplification 21  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE PCR - Primer non-target region Primers recognize specific region forming double strands DNA for polymerase binding -primer restricted amplification region of DNA is - only target region amplified PCR – Annealing Tm - Basic Melting Temperature (Tm) Calculations For sequences less than 14 nucleotides the formula is: Tm= (wA+xT) * 2 + (yG+zC) * 4 where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively. For sequences longer than 13 nucleotides, the equation used is P 46 4 Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC) - e.g. Calculate Tm for the following genes im 57 93 A. ~ 57 93. 57 52 bind LE X. 51. 78 20 64 9. + 41 * (4 + 7 - 16. 4)/(4 + 5+ 4 + 7) * = 53. 83 * 64 9 + 4 1 (6 + 4 16 4)/(6 + 4 + 6 + 4) X 51 78 -... =. 22  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE PCR – Thermocycler - DNA coping machine Genetically modification & Genetically modified organism (GMO) - Genetically modified organism (GMO) - GMO is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology. - Recombinant DNA technology use DNA molecules from different sources, which are combined into one molecule to create a new set of genes. This DNA is then transferred into an organism, giving it modified or novel genes. - GMOs are used in scientific research, drugs development and agriculture. GMF - common examples & advantages 23  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE GMF Testing in laboratory - most transgenic plants contain 3 elements: All set of genea contains in GM Food Remember # the example - most commercially available GM crop contain CaMV 35S promoter and NOS- terminator 24  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE GMF Testing in laboratory – PCR application - Testing GM tofu using PCR - amplify CaMV 35S region  to verify the GM identity - amplify lectin gene  to verify the food identity N - ↑A band > - make X from soy bean ↑ GM ↓ Central dogma of biology – classic view “The coded genetic information hard-wired into DNA is transcribed into individual transportable cassettes, composed of messenger RNA (mRNA); each mRNA cassette 25  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE contains the program for synthesis of a particular protein (or small number of proteins)." Gene expressions - Proteins  form cellular components, (supporting, cell adhesion molecules) biochemical components (enzymes, receptors), cells, tissues, organs … … Regulation of gene expressions - Natural / artificial - Toxic / non-toxic eg. ~) suppress - Drugs Hormones Toxicant 26 Environmental  BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE Central dogma of biology – modern view - New discoveries and exceptions The classic view reflects how molecular biology data are organized within the databases (e.g, by molecule type such as genomic DNA, mRNA, protein). However, many exceptions to this dogma are now known. (e.g. much of the DNA that does not encode prote ins is now known to encode various types of functional RNAs) Genes > - Proteins DNS that doesn't code for protein - RNA splicing - not all the transcribed DNA appear in the final mRNA - intron are removed same gene primary transcript transcript runn Nature mRNA XO 27  BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE Gene amplification – Reverse transcription-polymerase chain reaction (RT-PCR) - Reverse transcription - transcribe mRNA into complementary DNA (cDNA) using reverse transcriptase (RNA-dependent DNA polymerase) - RT-PCR – common technique for mRNA detection & quantitation - Only the -meaningful meaning and coding region of DNA are appear in the form mRNA which are translated in protein synthesis process - Detection of expressed genes is critically important in modern research mature Primers : Pligo-di , specific , random nexamers A Di - # yprime 28  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE RT-PCR - application - Detection of specific gene expression - e.g. M E E E ① RgIAVEGF ? Ely yuk & Dose response ? cendent der 7 ⑤ time lasting ! long EPA 29  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE Real-time quantitative reverse transcription PCR - qPCR - enables reliable detection and measurement of products generated during each cycle of PCR process (i.e., in real time) - reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles - the higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed per ⑦ - at amplified no directly existing gene lopy. ↑ material -- of fluorescence starting affect the intensity of signal being detected. 34 14 2 - signald there 478 how 30 2- lat beginning the have? many gone  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE ① qPCR – TaqMan® chemistry - TaqMan® chemistry (also known as “fluorogenic 5´ nuclease chemistry”) uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during each PCR cycle - 5’ exo-nuclease activity of special designed polymerase and Fluorescent Resonant Energy Transfer (FRET) makes it possible to detect PCR amplification PCR Synthesis/ elongate - Exo-Nuclease Activity of Taq Polymerase ↓ for - polymerase is adding bases to a growing chain of DNA; subsequently, close tyt > - the 5’ exo-nuclease activity of enzyme is removing DNA that is to transfer energy dye (red) downstream, impeding its’ capability to synthesize the new strand low energy far away -> keep energy in green - FRET (Fluorescent Resonance Energy Transfer) - when a high-energy dye is in close proximity to a low-energy dye, there will be a transfer of energy from high to low elongation probe L polymeras i , , I 1 #11211 m - detect the two color > - 5’ nuclease assay - - an oligonucleotide called a TaqMan® Probe is designed to anneal with a specific sequence of template between the forward and reverse primers - the probe sits in the path of the enzyme as it starts to copy DNA or cDNA; when the enzyme reaches the annealed probe the 5’ exonuclease activity of the enzyme cleaves the probe - when TaqMan® probe is intact and excited by a light source, the Reporter dye’s emission is suppressed by the Quencher dye as a result of the close proximity of the dyes - when the probe is cle aved by the 5’ nuclease activity of the enzyme, the distance between the Reporter and the Quencher increases causing the transfer of energy to stop; the fluorescent emissions of the 31  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE reporter increase and the quencher decrease - when the fluorescent signal Reporter increases to a detectable level it can be captured and displayed - the amount of reporter signal increase is proportional to the amount of PCK product being produced for a given sample qPCR - Amplification plot - Baseline - a reporter fluorescent signal is accumulating but is beneath the limits of detection of the instrument - ΔRn - an increment of fluorescent signal at each time point - Threshold - an arbitrary level of fluorescence chosen on the basis of the baseline variability. A signal that is detected above the threshold is considered a real signal that can be used to define the threshold cycle (Ct) for a sample. Threshold can be adjusted for each experiment so that it is in the region of exponential amplification across all plots. - Ct - defined as the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold 32  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE with sample blue 10 Copies generate the amplification linear cuvre after amplification 35 cycle why , ? yb now-Fi starting material d * Sgene sample copies no.it, P ↑ starting starting material material is abundant. Need more is mini. qPCR – CT calculation amplification. * draw a line ! How fold many the genes ? - e.g. calculate the fold change 004 x 2 - - = 2 = ?? Remember equation ! replicates (eg. U 3) A 13 5 B 12 = = = AverageOct for gene = the. , X Calculate &Oct (treatment-control) 12-13 5 = :. - (12 - 13. 5) 2 = 2. 93 33  BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE qPCR – SYBR® Green staining - a highly specific, double-stranded DNA (Minor Groove) binding dye, to detect PCR product as it accumulates during PCR cycles - detect all double-stranded DNA, including non-specific reaction products - binding of SYBR Green dye increase the intensity of the fluorescent emissions - working mechanisms - polymerase amplifies the target sequence, and found the double-stranded DNA working Compare - SYBR Green I dye binds to each new copy of double-stranded mechanism ①& DNA. Tagman - an increase in fluorescence intensity proportionate to the amount of PCR product produced SYBR 34  BIOL3046 PART I – Biomolecules and thei r detection D r. P YUE TaqMan chemistry vs. SYBR® Green staining TaqMan chemistry SYBR® Green staining Advantages High specificity - Specific Simple theory - used to hybridization between detect the probe and target is amplification of any required to generate double-stranded DNA fluorescent signal sequence Multi-detection - Probes No probe is needed can be labeled with different, distinguishable reporter dyes, which allows amplification of two distinct sequences in one reaction tube Cost effective - Post-PCR processing is not necessary Disadvantages Design and synthesis of False positive - SYBR specific probes is Green I dye binds to required for particular any double-stranded sequences. DNA, it can also bind to nonspecific double- stranded DNA sequences Compare Real time PCR vs. Traditional PCR - Limitations of End-Point PCR - Agarose gel electrophoresis is used for the end-point detection, but it is time consuming step - Poor precision - it based on size discrimination and the end point is variable from sample to sample, gels may not be able to resolve these variabilities in yield - Low resolution / sensitivity - agarose Gel - about 10-fold; Real-Time PCR - 2-fold change - Highly quantitative – EB dye (Traditional PCR), fluorescent dye (qPCR) - Efficiency – qPCR is automated - During PCR, some components are being consumed. This depletion will occur at different rates for each replicate. The reactions start to slow down and the PCR product is no longer being doubled at each cycle. This linear amplification can be seen in the linear phase of the reaction. The three samples begin to diverge in their quantities during the linear phase. - Each reaction will plateau at different point, due to the different reaction kinetics for each sample. These differences can be seen in the plateau phase, where is the detection point (end-point) of traditional PCR. 35  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE - The 3 replicate samples, which started out at the same quantity in the beginning of the reaction, reflect different quantities at the plateau phase. Since the samples are replicates they should have identical quantities. Therefore, it will be more precise to take measurements during the exponential phase, where the replicate samples are amplifying exponentially. - In qPCR, amplification phases can be viewed differently to assess the PCR phases - Logarithmic and Linear scale view. - Problems with detection in the Plateau phase of PCR - As stated earlier, the plateau region is the end-point of the reaction and is representative of the amount of product that you would see on Agarose Gels. All the replicates in the exponential phase are very tight in both the linear and logarithmic views. - In logarithmic view, the plateau for each reaction seems to occur in the same place, but this is solely due to the log scaling of the plot. - In line ar view, the reactions show a cle ar separation in the plateau phase; therefore, if the measurements were taken in the plateau phase, quantitation would be affected. 36  BIOL3046 PART I – Biomolecules and their detection Dr. P YUE high/no ??? -END- O V real-time vs Ri Lamp to Compare scan (role) ~ quick 37