Presentation on theme: "Genetic Technologies I should also acknowledge in advance all the people whose teaching material I’ve pinched off the web for these talks. http://www.stats.gla.ac.uk/~paulj/tech_genetics.ppt."— Presentation transcript:
1 Genetic TechnologiesI should also acknowledge in advance all the people whose teaching material I’ve pinched off the web for these talks.
2 Overview Why learn about genetic technologies? The molecular geneticist’s toolkitGenetic markersMicroarray assaysTelomeresRNA interference (RNAi)This talk covers a very large field, so the aim is not to give a detailed understanding of particular techniques, but first to understand the basic tools that lie behind the techniques used to generate molecular genetic data, and then to illustrate them by giving examples of the most common techniques.
3 Why learn about genetic technologies? In general, anyone doing data analysis would agree that it’s important to understand the data we’re analysing. This is obviously true whether we’re analysing blood pressures, the amount people smoke, blood cholesterol levels or genotypes. We obviously need to know how data is produced before we can even decide what kind of analysis is appropriate. However, a more detailed level of knowledge can also help us as assess the data’s reliability, and how errors might affect the conclusions of our analysis. The aim of this talk is to understand the processes behind the generation of genetic data. Not so much the biological processes, because its assumed that we’ll understand these before we plan and carry our the data analysis, but the laboratory processes.
4 Why learn about genetic technologies? We need to understand the processes that generated the dataUnderstanding of biology obviously necessaryUnderstanding of lab techniques will enhance our ability to assess data reliabilityErrors in any measurement can lead to loss of power or biasSome genetic analyses are particularly sensitive to error becausethey depend on the level of identity between DNA sequences shared by relativesthe more data is collected, the greater the chance of false differencesBefore we can analyse data we need to understand the process that generated it
5 Why learn about genetic technologies? IndividualGenotypeA177, 179B179, 179If we take this example of two rows of genotype data. These data come from microsatellite markers, which I’ll introduce properly later but for the moment we just need to know that they record the length of a particular bit of DNA. Here we can see two individuals, A and B, with different genotypes, A has etc. We might be interested in this marker because we know that, in a particular family, allele 177 is close on the genome to a disease allele, so we’re using this microsatellite locus as a marker of this disease allele.What is the probability that the observed genotype is wrong?Is this probability the same for all observed genotypes?What impact will a realistic range of errors have on power?
7 Most genetic technologies are based on four properties of DNA DNA can be cut at specific sites (motifs) by restriction enzymesDifferent lengths of DNA can be size-separated by gel electrophoresisA single strand of DNA will stick to its complement (hybridisation)DNA can copied by a polymerase enzymeDNA sequencingPolymerase chain reaction (PCR)Some of these properties apply to RNA as well
8 DNA can be cut at specific sites (motifs) by an enzyme Restriction enzymes cut double-stranded DNA at specific sequences (motifs)E.g. the enzyme Sau3AI cuts at the sequence GATCMost recognition sites are palindromes: e.g. the reverse complement of GATC is GATCRestriction enzymes evolved as defence against foreign DNASau3AIGATCCTAG
9 DNA can be cut at specific sites (motifs) by an enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGALet’s say we have a sample that consists of a short length of DNA that we happen to be interested in because it’s involved in causing a disease. We might have obtained this piece of DNA by the polymerase chain reaction, which I won’t go into just now but we’ll come back to that later.
10 DNA can be cut at specific sites (motifs) by an enzyme Sau3AIGATCCTAGACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
11 DNA can be cut at specific sites (motifs) by an enzyme ACTGTCGATGTCGTCGTCGTAGCTGCT GATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAG CATCGATCGASau3AIGATCCTAG
12 DNA can be cut at specific sites (motifs) by an enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTTGACAGCTACAGCAGCAGCATCGACGACTAGGATCGTAGCTAGCTCATCGATCGAACTGTCGATGTCGTCGTCGTAGCTGCTGATGACAGCTACAGCAGCAGCATCGACGACTTCGTAGCTAGCTAGCATCGATCGAWe’ll see why this is useful in a minutbut first we need to km]now about another technique
13 Different lengths of DNA can be separated by gel electrophoresis DNA is negatively charged and will move through a gel matrix towards a positive electrodeShorter lengths move fasterYou can’t actually see the DNA while it’s running through the gel. Generally the DNA will be made visible after the gel has finished either by a DNA specific stain, or because it had previously been marked with a radioactive or fluorescent label. Another very useful property of this technique is that once a piece of DNA has been separated on a gel, it can be recovered unharmed for further analysis, and can even be inserted into a living organism (often a bacterium).
14 Different lengths of DNA can be separated by gel electrophoresis DNA is negatively charged and will move through a gel matrix towards a positive electrodeShorter lengths move fasterYou can’t actually see the DNA while it’s running through the gel. Generally the DNA will be made visible after the gel has finished either by a DNA specific stain, or because it had previously been marked with a radioactive or fluorescent label. Another very useful property of this technique is that once a piece of DNA has been separated on a gel, it can be recovered unharmed for further analysis, and can even be inserted into a living organism (often a bacterium).
15 Different lengths of DNA can be separated by gel electrophoresis Slow: 41 bpACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGAMedium: 27 bpACTGTCGATGTCGTCGTCGTAGCTGCTTGACAGCTACAGCAGCAGCATCGACGACTAGFast: 10 bpGATCGTAGCTAGCTCATCGATCGAFMS
16 Different lengths of DNA can be separated by gel electrophoresis Recessive disease allele D is cut by Sma3AI:ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGAHealthy H allele is not cut:ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGAFMSHHHDDD
17 Different lengths of DNA can be separated by gel electrophoresis MSHHHDDD
18 A single strand of DNA will stick to its complement ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
19 A single strand of DNA will stick to its complement ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
20 A single strand of DNA will stick to its complement ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
21 A single strand of DNA will stick to its complement ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
22 A single strand of DNA will stick to its complement Fragment frequencyFlourescenceThis property is useful because we almost always want to focus on a particular part or parts of the human genome, for example a putative disease gene, or telomeres. If we look back at our earlier example where we were analysing a short fragment of DNA that we got by the polymerase chain reaction that carried a disease-linked mutation. PCR is one way of looking at a particular bit of DNA, and it depends on this property of specific hybridisation. An earlier method, one that is still in use, is Southern blotting, which we’ll look at now. We don’t start out from the point of having a convenient manageable piece of DNA that we’re interested in, we have whole chromosomes, which are too big to practically separate with a gel. We can cut them up using a restriction enzyme, but if we run that through a gelFragment length in bp
23 A single strand of DNA will stick to its complement
24 A single strand of DNA will stick to its complement Southern blotting (named after Ed Southern)
25 A single strand of DNA will stick to its complement Southern blotting (named after Ed Southern)
26 A single strand of DNA will stick to its complement
27 A single strand of DNA will stick to its complement
28 A single strand of DNA will stick to its complement This technique allows you pick out specific DNA sequences. It’s main drawbacks are that it’s slow and therefore expensive, and requires a fairly large amount of DNA.
29 DNA can copied by a polymerase enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
30 DNA can copied by a polymerase enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGAAGTGCAAGCTDNA polymeraseGGAAGAGTTCTCCCAGTAAG
31 DNA can copied by a polymerase enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGAAGTGCAAGCTDNA polymeraseGGAAGANow lets say we add to the mix a small number of bases that are modified in two ways: (1) they cause the chain to stop growing and (2) each letter has a separate dye attached.GTTCTCCCAGTAAG
32 DNA can copied by a polymerase enzyme ACTGTCGATGTCGT
33 DNA can copied by a polymerase enzyme ACTGTACTGTCGATACTGTCGATGTACTGTCGATGTCGTACTGTCGATGTCGTCGTACTGTCGATGTCGTCGTCGTACTGTCGATGTCGTCGTCGTAGCTACTGTCGATGTCGTCGTCGTAGCTGCTACTGTCGATGTCGTCGTCGTAGCTGCTGATACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
34 DNA can copied by a polymerase enzyme ACTGTCGATGTACTGTCGATGACTGTCGATACTGTCGAACTGTCGACTGTCACTGTTGACTimeFluorescenceT C G A T G T etcFluorescenceTime
37 DNA can copied by a polymerase enzyme Add original paper
38 DNA can copied by a polymerase enzyme Polymerase chain reaction (PCR)A method for producing large (and therefore analysable) quantities of a specific region of DNA from tiny quantitiesPCR works by doubling the quantity of the target sequence through repeated cycles of separation and synthesis of DNA strands
42 DNA can copied by a polymerase enzyme Like southern blotting, PCR allows you to examine a single specific site on the genome. In southern blotting you get the specificity from the bind of the probe to the template. In PCR the job of the probe is done by the primers, which pick out specific sequences flanking the area of interest. One of the main advantages of PCR is that you only need a tiny amount of DNA for each reaction, so you do 100s or 1000s of assays on a single sample, whereas southern blots require relatively quantities. E.g. a blood sample might have enough DNA for one blot but 1000s of PCRs. PCR is also much easier to automate and scale up, and therefore much cheaper.
43 DNA can copied by a polymerase enzyme One of the
44 DNA can copied by a polymerase enzyme In the words of its inventor, Kary Mullis…PCR can generate 100 billion copies from a single DNA molecule in an afternoonPCR is easy to executeThe DNA sample can be pure, or it can be a minute part of an extremely complex mixture of biological materialsThe DNA may come froma hospital tissue specimena single human haira drop of dried blood at the scene of a crimethe tissues of a mummified braina 40,000-year-old wooly mammoth frozen in a glacier.In the words of its inventor, PCR can…This list is interesting because the quality of the data you get out of PCR depends very much on the quality and quantity of DNA that goes in. Very high error rates can be associated with using low quality and quantity DNA, although it’s less well appreciated that certain kinds of errors can result from using too much DNA in a PCR reaction.
45 DNA can copied by a polymerase enzyme The invention of PCR had a huge impact on genetics, and won the nobel prize in Chemistry for its inventor, the rather eccentric Kary Mullis.
46 The molecular geneticist’s toolkit Specific DNA-cutting restriction enzymesDNA size separation by gel electrophoresisHybridisation using labelled DNA probesSynthesis of DNA using DNA polymerase (PCR)(and ligase for re-joining cut ends)With these tools we construct most genetic assays.
47 Genetic markersThe first class of techniques I’m going to talk about is genetic markers.
48 Genetic markers What are they? Variable sites in the genome What are their uses?Finding disease genesTesting/estimating relationshipsStudying population differencesWhat do they do?
49 Eye colour Phenotype Genotype Brown eyes BB or Bb Blue eyes bb A genetic marker doesn’t have to be hi tech, and it doesn’t even have to be a direct assay of DNA. Any trait that is under strong genetic control could be used. You could use eye colour as a dominant marker.
50 ABO blood group Phenotype Genotype AB A AA or AO B BB or BO O OO The ABO blood group has been used as a genetic marker for about 90 years. It is a partially dominant marker.Obviously eye colour and blood group have their disadvantages as genetic markers. Each is dominant to some extent, so some information is hidden. Each is also a single marker. If we want to search across the whole genome for a gene that contributes to disease, we need markers that are spread across the whole genome. They also don’t directly assay the DNA mutation behind the change, so there’s the possibility firstly that we’re missing some of the underlying DNA variation, and that there isn’t a perfect correlation between genotype and phenotype (for example, what do we do with green eyes?).
51 The ideal genetic marker CodominantHigh diversityFrequent across whole genomeEasy to findEasy to assaySo our shopping list for the perfect marker would look something like this:
52 Modern genetic markers: SNPs SNPs are single nucleotide polymorphismsUsually biallelic, and one allele is usually rareCan be protein-coding or notThis example is a T/G SNP. An individual can be TT, TG, GGHealthy allele A is cut by Sma3AI:ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGARecessive disease B allele is not cut:ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGAThey can have up to 4 alleles, but almost all of them have 2, simply because mutations are very rare and the chance of two occuring at the same base, and both reaching detectable frequency in the population is very small. W measure the diversity of a marker by probability of any two alleles chosen at random from the population being different.
61 Modern genetic markers: microsatellites Microsatellites are short tandem repeats (STR, also SSR)Usually high diversityUsually not in protein coding sequenceThis example is an (AC)n repeat; a genotype is usually written n,nWith k alleles there are k(k+1)/2 possible unordered genotypesACTGTCGACACACACACACACGCTAGCT (AC)7TGACAGCTGTGTGTGTGTGTGCGATCGAACTGTCGACACACACACACACACGCTAGCT (AC)8TGACAGCTGTGTGTGTGTGTGTGCGATCGAACTGTCGACACACACACACACACACACGCTAGCT (AC)10TGACAGCTGTGTGTGTGTGTGTGTGTGCGATCGAACTGTCGACACACACACACACACACACACACGCTAGCT (AC)12TGACAGCTGTGTGTGTGTGTGTGTGTGTGTGCGATCGA789127,77,88,87,98,99,97,128,129,1212,12Diversity is much higher in microsatellites as they usually have many alleles due to having much higher mutation rates. Diversity tends to be higher than 0.5 and is generally about 0.75.
66 Microsatellites versus SNPs CodominantYesDiversityHighLowFrequent10,000s3 millionEasy to assayEasy to findNoNo, but…Other advantages of SNPs: they are very simple and they’re all pretty similar, which makes them easier to automate; having only 2 states reduces the possibility of error compared with microsatellites.
67 Uses of SNPs and microsatellites The HapMap project has discovered millions of SNPsTheir high density in the genome makes them ideal for association studies, where markers very close to disease genes are requiredMicrosatellitesMore suitable for family-based studies, where high variation is valuable and lower levels of resolution are requiredPopulation association studies: need close markers because we’re looking for associations between disease genes and markers that have persisted over hundreds of generationsFamily: association just needs to survive one generationThere is crossoverSNPs may win as they become cheaper and faster to genotype
68 Overview Why learn about genetic technologies? The molecular geneticist’s toolkitGenetic markersMicroarraysTelomeresRNA interference (RNAi)This talk covers a very large field, so the aim is not to give a detailed understanding of particular techniques, but first to understand the basic tools that lie behind the techniques used to generate molecular genetic data, and then to illustrate them by giving examples of the most common techniques.
69 The molecular geneticist’s toolkit Specific DNA-cutting restriction enzymesDNA size separation by gel electrophoresisHybridisation using labelled DNA probesSynthesis of DNA using DNA polymerase (PCR)(and ligase for re-joining cut ends)With these tools we construct most genetic assays.
70 Overview Why learn about genetic technologies? The molecular geneticist’s toolkitGenetic markersMicroarraysTelomeresRNA interference (RNAi)This talk covers a very large field, so the aim is not to give a detailed understanding of particular techniques, but first to understand the basic tools that lie behind the techniques used to generate molecular genetic data, and then to illustrate them by giving examples of the most common techniques.
71 MicroarraysMicroarrays are a way of telling which genes are switched on and to what degree…
72 Gene expression Transcription: DNA gene → mRNA in nucleus Translation: mRNA → proteinin cytoplasmMicroarrays use mRNA as a marker of gene expressionNucleusCytoplasm
73 What are microarrays?A microarray is a DNA “chip” which holds 1000s of different DNA sequencesEach DNA sequence might represent a different geneMicroarrays are useful for measuring differences in gene expression between two cell typesThey can also be used to study chromosomal aberrations in cancer cells
74 Principles behind microarray analysis Almost every body cell contains all ~25,000 genesOnly a fraction is switched on (expressed) at any time in any cell typeGene expression involves the production of specific messenger RNA (mRNA)Presence and quantity of mRNA can be detected by hybridisation to known RNA (or DNA) sequences…as a surrogate for the level of gene expression. Why would you want to know this?
75 What can microarray analysis tell us? Which genes are involved indisease?drug response?Which genes areswitched off/underexpressed?switched on/overexpressed?For example,
76 Before microarrays: northern blotting Extract all the mRNA from a cellSize-separate it through a gelMeasure level of expression using a probe made from your gene of interest
77 Northern blotting: still useful for single-gene studies
81 Microarrays can be used to diagnose and stage tumours, and to find genes involved in tumorigenesis Copy number changes are common in tumoursLoss or duplication of a gene can be a critical stage in tumour developmentFrequency plot of copy number alterations nFeiggautrivee 1 tumors tions in ER positive andFrequency plot of copy number alterations in ER positiveand negative tumors. The top two panels show thefrequency of gains, indicated by the green bars ranging from 0to 1, and losses, indicated by the red bars ranging from 0 to -1, in 62 sporadic breast tumors for each clone. The bottompanel displays the magnitude of the t-statistic for each clonecomputed based on the smoothed data as described in theMethods. The horizontal dotted lines indicate the statisticcut-off corresponding to the FDR-adjusted p-value of 0.05(blue) and 0.1 (green).ChromosomeBMC Cancer 2006, 6:96
82 Problems of microarray analysis Gene expression ≠ mRNA concentrationEasy to do, difficult to interpretStandardisation between labsLots of noise, lots of genes (parameters)e.g. p = 10,000low sample sizee.g. n = 3
84 Telomeres and telomerase Telomeres are repetitive DNA sequences at the ends of chromosomesThey protect the ends of the chromosome from DNA repair mechanismsIn somatic cells they shorten at every cell division, leading to agingIn germ cells they are re-synthesised by the enzyme telomeraseCentromereEvery time a cell multiplies to make two new cells, special zones at the ends of its chromosomes, called telomeres, become shorter. Once the telomeres reach a certain length, the cell stops dividing and eventually dies. The only cells to escape this fate are those that divide to make eggs and sperm. In these cells, a substance called telomerase builds the telomeres up again, so they remain the same length.Telomere
85 Why do we need telomeres? At every cell division each chromosome must be replicatedDNA is synthesised in one direction onlyThe “lagging strand” is synthesised “backwards” in 100–200 bp chunksSpend some time on this. Be sure to mention leading and lagging strands.
86 Leading strandThis isn’t a problem for the leading strand…
87 Lagging strand…but 100–200 bp of single stranded DNA are left hanging at the end of the lagging strand, and are lost.So at every cell division bases are lost.
92 Health implications of telomere shortening: cancer Cancer tumour cells divide excessively, and will die unless they activate telomeraseTelomerase activation is an important step in many cancer cell typesTelomere length can be used to diagnose tumoursTelomerase is a potential target of cancer therapyWhat role do telomeres play in cancer?As a cell begins to become cancerous, it divides more often, and its telomeres become very short. If its telomeres get too short, the cell may die. It can escape this fate by becoming a cancer cell and activating an enzyme called telomerase, which prevents the telomeres from getting even shorter.Studies have found shortened telomeres in many cancers, including pancreatic, bone, prostate, bladder, lung, kidney, and head and neck.Measuring telomerase may be a new way to detect cancer. If scientists can learn how to stop telomerase, they might be able to fight cancer by making cancer cells age and die. In one experiment, researchers blocked telomerase activity in human breast and prostate cancer cells growing in the laboratory, prompting the tumor cells to die. But there are risks. Blocking telomerase could impair fertility, wound healing, and production of blood cells and immune system cells.
93 Measuring telomeres Two principal methods Southern blotting Quantitative PCR (qPCR)
94 Measuring telomeresTRF = telomere restriction fragment length
95 RNA interference (RNAi) The first class of techniques I’m going to talk about is genetic markers.
96 What is RNAi?Generally genes are studied through the effects of knockout mutations in particular experimental organismsRNAi is a quick and easy technique for reducing gene function without the necessity of generating mutants that can be applied to any organismIt has the potential to treat diseases caused by over-expression of genesThe discoverers, Andrew Fire and Craig Mello, have just won the Nobel prize (announced 2 October).
97 Principles of RNA interference (RNAi) Injection of double-stranded RNA (dsRNA) complementary to a gene silences gene expression bydestruction of mRNAtranscriptional silencingstopping protein synthesisGene expression can be switched off in specific tissues or cells by the injection of specific dsRNATakes advantage of endogenous defences against double-stranded RNA viruses??????
98 RNAiRNAi is a natural phenomenon, which probably evolved as a defence against dsRNA viruses, and is also involved in the regulation of gene expression.
99 Fascinating facts: each cell contains about 2 m of DNA, divided into 46 chromosomes (so each chromosome is on average 5 cm long, that means it’s 10,000 times longer than the width of the nucleus). Clearly this is an organisational problem.
100 Uses of RNAiInvestigating role of genes by knocking down (not out) gene expression in specific tissues at specific developmental stagesPotential use in gene therapymacular degeneration: two phase I trials currently under waytherapies being developed for HIV, hepatitis, cancers
101 Limitations of RNAiTarget specificity: how do you know the dsRNA isn’t interfering with other genes?Interpretation of resultsRisks for gene therapyFunction isn’t knocked out, it’s reducedKnockdown may not reveal gene functionMight not give therapeutic effectGene therapy dead mice look up liver problem