Lecture 10: PCR and Sequencing PDF

LF130 Cellular and Molecular Biology Lecture 10, 2024. DNA sequencing and PCR Part 1. Techniques Dr Robert Spooner  Summary, to date 5’...

LF130 Cellular and Molecular Biology Lecture 10, 2024. DNA sequencing and PCR Part 1. Techniques Dr Robert Spooner  Summary, to date 5’ 3’ 5’ 5’ 3’ 3’ 5’ Confirmation of the Watson-Crick predictions: DNA replication is semi- conservative DNA strands are anti-parallel Watson-Crick base pairing underlies both of these New DNA is synthesised in the 5’ → 3’ direction: DNA synthesis is semi-continuous, with a leading a lagging strand DNA sequencing - order of nucleotides in a piece of DNA. Walter Gilbert, Biology Labs, Harvard. Partial chemical degradation of radiolabelled DNA Frederick Sanger, MRC Labs, Cambridge, 1970s. Enzyme-mediated incorporation of dideoxynucleotides into newly- replicated DNA  Sanger sequencing - dideoxynucleotides O- O- O- - O P O - O P O - O P O Relies on the incorporation of O O O dideoxynucleotides - O P O - O P O - O P O into newly replicated DNA O O O - O P O - O P O - O P O O O O CH2 O base CH2 O base CH2 O base H H H H H H H H H H H H OH OH OH H H H Ribonucleoside 2’- 2’,3’- triphosphate, Deoxyribonucleosid Dideoxyribonucleoside  A dideoxynucleotide is a TERMINATOR 3 ’ 5 3 ’ ’ ddNTP 3 5 5 3 ’ ’ ’ ’ 3 ’ 3 5 5 3 ’ ’ ’ ’ 3 5 5 ’ ’ ’ 3 5 5 3 ’ ’ ’ ’ ddNTPs have no 3’-OH group. When incorporated 3 5 ’ ’ into DNA, no more nucleotides can be added: Normal extension using ddNTPs are CHAIN TERMINATORS dNTPs  Sanger’s dideoxy sequencing method: the principle Add: a single stranded template DNA a primer complementary to part of this template DNA polymerase a pool of normal deoxynucleotides (dATP, dTTP, DGTP and dCTP) a small proportion of radioactively-labelled ddATP 5’-CTAGGACTTCGAAGTC-3’ 3’-GATCCTGAAGCTTCAGAGCACAGCTATCGCGA-5’ 5’-CTAGGACTTCGAAGTCTCGTGTCGATAGCGCT-3’ 5’-CTAGGACTTCGAAGTCTCGTGTCGATA-3’ reaction products 5’-CTAGGACTTCGAAGTCTCGTGTCGA-3’ Some newly synthesized molecules will get a ddATP in each place that there is a T in the template DNA. The result is a nested set of new DNA molecules each of which ends in a ddA. Sanger’s dideoxy sequencing method: the practice 5’-CTAGGACTTCGAAGTC-3’ ① template + primer + DNA Pol + 3’-GATCCTGAAGCTTCAGAGCACAGCTATCGCGA-5’ dNTPs ② add appropriate ddNTP GATC 3’ + ddG + + + ddC ddA ddT 5’PTCGTGTCGATAGCG 5’PTCGTGTCGATA 5’PTCGTGTCGATAGCGCT 5’PTCGTGTCGATAGCGC 5’PTCGTGTCGATAG 5’PTCGTGTCGA 5’PTCGTGTCGAT 5’PTCGTGTCGATAGC 5’PTCGTGTCG 5’PTCGTGT 5’PTCGTGTC 5’PTCGTG 5’PTCGT 5’PTC 5’PTCG 5’PT ③Separate the nested fragments on the basis of size by electrophoresis. 5’  Automated dideoxy sequencing ① template + primer + DNA 5’-CTAGGACTTCGAAGTC-3’ Pol + all dNTPs + all 3’-GATCCTGAAGCTTCAGAGCACAGCTATCGCGA-5’ fluorescent ddNTPs in ONE tube 5’PTCGTGTCGATAGCGCT ②The dye present in each synthesized 5’PTCGTGTCGATAGCGC 5’PTCGTGTCGATAGCG fragment corresponds to the dye attached to 5’PTCGTGTCGATAGC + ddG 5’PTCGTGTCGATAG the dideoxynucleotide that terminated the 5’PTCGTGTCGATA synthesis of that particular fragment. + 5’PTCGTGTCGAT 5’PTCGTGTCGA ddA + 5’PTCGTGTCG 5’PTCGTGTC ③ Pass nested products through an +ddT ddC 5’PTCGTGT 5’PTCGTG electrophoretic system and read with 5’PTCGT lasers 5’PTCG 5’PTC 5’ T C G T G T C G A T A G C G C T 3’ 5’PT  What is the link between…. Good evening, Doctor … a glowing green raccoon that said …. … a potent recreational drug with psychological effects including synaesthesia, an altered sense of time and spiritual experiences … … claims that climate change and the connection between HIV and … and a Nobel Prize … … belief in astrology AIDS are … … ? conspiracy theories … Kary Mullis and Polymerase chain reaction (PCR) One of the most powerful tools in molecular biology. Invented by Kary Mullis in 1983, resulting in his Nobel Prize in Chemistry. In essence, PCR acts as a “copying machine” for DNA. It is a tool to amplify a specific DNA sequence  A brief history… 1953 Watson and Crick: DNA model. 1957 Kornberg: first DNA polymerase. 1960s Har Gobind Khorana started deciphering the genetic code (see later). 1969 Thomas Brock isolated a bacterium, Thermus aquaticus, from a hot spring in Yellowstone National Park. 1971 Khorana's group suggested that a template-primer-polymerase system could be used for copying DNA … and using two primers should lead to replication of a specific fragment of DNA (but did not show results). 1976 Taq polymerase was isolated from T. aquaticus.  Kary Mullis put them all together The components: Template dsDNA with a target area, and DNA sequence knowledge. Two SPECIFIC oligodeoxynucleotide primers (typically 20-24 mers) which can hybridise at each end of the target sequence. dNTPs. Buffer and MgCl2 and Taq polymerase primer A TARGET DNA 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ primer B  Why is Taq polymerase useful? Thermus aquaticus, a Gram negative rod, was isolated from this spring, from the outflow (70°C) T. aquaticus thrives at ~70°C but can survive at temperatures between 50°C to 80°C. PCR is possible because of the thermal stability of Taq DNA  How does PCR work? The method: Amplification of the target DNA is achieved by repeated cycles of: (a)heating (94°C) to denature the target DNA. Taq polymerase is NOT denatured by this, even after multiple cycles. (b) hybridisation of primers, one to each strand (45-65°C) (c) extension of the primers by Taq DNA polymerase (72°C) Typically ~30 cycles are performed. Care over temperature control is essential. PCR machines are programmable for use with many different cycling parameters and very rapidly change the temperature between the various stages of the PCR process.  How does it work? Cycle 1 Target 5’ 3’ 3’ 5’ Start: one ds copy of what you wish to amplify  How does it work? Cycle 1 Target 5’ 3’ Heat at 94C, 1-2 minutes, to separate the strands Cool to ~50C to allow ‘primers’ to bind 3’ 5’ Start: one ds copy of what you wish to amplify  How does it work? Cycle 1 Target 5’ 3’ Heat at 94C, 1-2 minutes, to separate the strands Cool to ~50C to allow ‘primers’ to bind Heat to 72C for 1-2 minutes to allow Taq polymerase to ‘extend’ the primers 3’ 5’ Start: one ds copy of what you wish to amplify End: you now have two copies of what you want  How does it work? Cycle 2 Target 5’ 3’ 3’ 5’ How does it work? Cycle 2 Target How does it work? Cycle 2 Target How does it work? Cycle 3 Target How does it work? Cycle 3 Target How does it work? Cycle 3 Target How does it work? Cycle 3 Target The sequence of this is specified by the target and the primers used The sequence of this is specified by the target and the primers used And the process is repeated about 30 times, automated One cycle: 2 copies 1.2x10 9 1.20E+09 Two cycles: 4 copies Three cycles: 8 copies 1x10 9 1.00E+09. number of copies. 8x10 8 8.00E+08. number of copies Ten cycles: 1024 copies. 6x10 8 6.00E+08.. 4x10 4.00E+08 8 Twenty cycles: >1 million copies 2.00E+08 2x10 8.. 0.00E+00 0 0 5 10 15 20 25 30. Thirty cycles: > 1000 million PCR cycles copies So, now all we have to do is visualize the PCR reactions … The technique is agarose gel electrophoresis M R1 R2 - The reactions are loaded into the wells of a slab gel made of agarose. An electric current is applied. DNA is negatively charged, so it migrates to the positive terminal. The gel contains a dye that binds DNA, so the DNA will fluoresce under ultraviolet light + Large fragments migrate SLOWLY: small fragments QUICKLY So, now all we have to do is visualize the PCR reactions … The technique is agarose gel electrophoresis M R1 R2 - +  Examining PCR reactions number of PCR cycles M - 0 1 2 4 6 8 10 15 20 30 So, from tiny amounts of DNA we can amplify large visible amounts. 1000 Size (bp) 400 Starting After 30 material: cycles: undetectable clearly visible  PCR has both specificity and sensitivity ① Specificity is provided by the primers: primerA 3’ 5’ 5’ 3’ target 3’ 5’ 5’ 3’ primerB They are complementary to opposite strands with their 3’ ends pointing towards each other. Complimentary: ‘Ooh, you do look lovely’. Complementary: making ‘whole’, in this case relating to specific base pairing between strands of a DNA or an RNA molecule. ② Sensitivity: one target molecule can be amplified to > 109 molecules in just a few hours, because a product formed in one cycle becomes a template for the next.  Applications of PCR Amplify tiny amounts of DNA to obtain enough for analysis by almost any of the usual molecular biological techniques, including DNA sequencing, cloning etc. Analyse small samples for medical-genetic purposes, from relatively non-invasive sampling, including cells scraped from inside cheek, or single drop of blood. Analyse tiny explants of tissue such as chorionic villus samples (will form placenta) in the first trimester of pregnancy, for pre-natal genetic screening. And to follow, in greater detail: Amplify archaeological and ancient DNA sources. Use PCR to alter a DNA sequence. Forensic applications using blood, semen, hair, human cells on cigarette butts, etc.  LF130 Cellular and Molecular Biology Lecture 10, 2024. DNA sequencing and PCR Part 2. Applications Dr Robert Spooner  Applications of PCR (1): Making specific mutations Mutations can be introduced into amplified DNA by engineering one or more mismatches in a primer close to or at its 5’ end. 5’ ACATGACCA… …CTATGCATG 3’ 3’ Target DNA duplex 5’ TGTACTGGT… …GATACGTAC 5’ ACATGACCA… …CTATGCATG 3’ GATACGTAC Denaturation and GCATGACCA primer binding 3’ 5’ TGTACTGGT… …GATACGTAC 5’ GCATGACCA… …CTATGCATG 3’ 3’ 5’ Final mutated CGTACTGGT… …GATACGTAC product Draw this out, each step, and see if you get the same final mutated product: if you do, you understand this … and you need to understand this to understand ‘Techniques 2’  How wide is this gap? Applications of PCR (2): sequencing of archaic hominin genomes Sequencing of archaic hominin genomes Green et al., 2010. Science 328:710-722 Nuclear DNA was amplified from fossil Neanderthal bones from the Vindija site (Croatia) and compared with DNA from modern humans. CONCLUSIONS: Europeans and Asians have ~ 4-5% of their genes derived from Neanderthals. Gene flow can be detected from Neanderthals into modern humans … but not the reverse, and no mtDNA gene flow has been detected. This suggests male Neanderthal and female human couplings. UPDATE: Later revision. Europeans have  Archaic hominin genomes: the story is even richer … and a finger bone and a couple of toe bones: their DNA has been sequenced. Subsequently, Denisovan Two teeth … fossils also found in Tibet and Laos. N D AFRICA EURASIA 0 H. sapiens H. floresiensis 0.4 … found in H. heidelbergensis H. erectus Million years ago this cave … 0.8 H. antecessor 1.2 … here… N Neanderthal 1.6 D Denisovan H. erectus 2.0  Archaic hominin genomes: and in 2011… … another type of human was found, even though there are NO hard fossils (no bones, no teeth) so we can’t even guess what they looked like. But they left a genetic signature in some African populations – a DNA fossil. And in 2018: a Neanderthal: Denisovan hybrid … 2021 – a brand new Eurasian human ? N D ? ? ‘Dragon Man’ 0 H. sapiens 2022: hints of another unknown genetic signature in Melanesians H. floresiensis 0.4 H. heidelbergensis H. erectus Million years ago N Neanderthal 0.8 D Denisovan H. antecessor 1.2 ? Who were they? DNA amplification and H. erectus 1.6 sequencing technologies are re- writing our origins. 2.0 AFRICA EURASIA MELANESIA If we can identify DNA from a 40,000 year-old Neanderthal … … can we identify living  If we can identify DNA from a 40,000 year-old Neanderthal … The Lord of the Manor is found dead …  Applications of PCR (3): Forensic science (‘forensic’ means ‘legal’) The size of a blood sample required for blood-type analysis PCR amplifies target DNA, so very small samples can be processed. DNA profiling. 1984, Sir Alec Jeffreys: VNTRs can be used to identify people. A Variable Number Tandem Repeat (or VNTR) is a run of short repeated nucleotide sequence (each typically 3-5 bp). These can be found on many chromosomes, and often show variations in length between individuals. Copley Medal Note: VNTRs are now called STRs (single tandem  Each STR acts as an inherited allele. The number of repeats is highly variable in the population and hence individuals will usually inherit a different variant of each STR locus from their mother and father. - chromosomes Homologous paternal PCR maternal + Primers are designed to bind The different sized fragments specifically upstream and can be separated downstream of an STR electrophoretically Thus, an STR can be used for personal or parental identification. The Lord of the Manor meets his end by cyanide … Forensic samples (epithelial cells) are collected from his pill box … Whodunnit ?  The prime suspects … The Lady The Mistress The Butler About to lose her Having a secret affair … the title through divorce. with the butler … undeclared illegitimate son of the Buccal swabs are taken from the three Lord. prime suspects  A single STR has little discriminatory power The Mistress The Lady The Mistress The Butler The Pillbox The Lord The Pillbox The Butler The Lady The Lord STR1 number of repeats 30 25 20 A single STR may be shared by unrelated 15 people. 10 The Lady is off the hook. She’s innocent! 5 It’s either the butler or the mistress.  STR analysis in practice CODIS: Combined DNA Index System, operated by the FBI Typically, a multiplex PCR is performed (multiple reactions in one tube) using specific primers for 13 different STRs on different chromosomes. D2S1338 D3S1358 THO1 D8S1179 D5S818 VWA D13S317 D16S539 D18S51 FGA D7S820 D21S11 CSF1PO There is also a European standard set: Together the US and European sets can (probably) identify EVERY person in the world (except identical twins), as long as X/Y status is included. X and Y chromosomes are identified in a separate reaction.  Using multiple STRs increases discrimination The Mistress The Pillbox The Butler The Lady The Lord VNTR2 WHODUNNI number of repeats 30 25 20 VNTR3 15 10 Yes!T?The butler 5  And where are they now? The Mistress The Pillbox The Lady The Mistress The Butler The Pillbox The Butler The Lady The Lord VNTR2 number of repeats 30 25 20 VNTR3 15 10 5 Inherited. Died alone Banged up for Exhibit in the with 42 cats. life. Black Museum.