Using Molecular Biology

Question 1

What are the uses from Molecular Biological tools?

Answer 1

• Molecular biological tools provide information about DNA and proteins
• Detection of the presence and distribution of genes, proteins, organisms in the environment
• Understand the molecular basis of interactions (eg symbiosis)
• Monitor disease
• Track and understand important
  
  • processes eg photosynthesis
  • Ecosystems eg deep sea / coral reefs
  • Impacts eg pollution and conservation
• Track and understand important development - find out whether photosynthesis is happening

Question 2

What did Carl Woese develop as a way to study evolutionary relationships of bacteria?

Answer 2

• DNA fingerprinting of bacteria by sequencing the 16S rRNA gene
• Showed the existence of Archaea in 1977
• Current database is tens of thousands of species
• Carl Woese started to look at evolutionary relationship between bacteria. Good place to start to look as all living organisms have ribosomes.
• Carl Woose discovered the 3rd branch of life - Archea.

Question 3

What is a ribosome made up of?

Answer 3

• Large ribosome made of a large subunit and a small subunit
  
  • Made up of proteins and RNA molecules
• Large ribosome made of 34 large subunits and 21 small subunits
  
  • Made up of proteins and RNA molecules

Question 4

Who was awarded the nobel proze for for his research on colloids and proteins using the ultracentrifuge, and what did they do?

Answer 4

Svedberg used centrifugal force to mimic the effects of gravity on them. His first ultracentrifuge, completed in 1924, was capable of generating a centrifugal force up to 5,000 times the force of gravity. Later versions generated hundreds of thousands of times the force of gravity. Svedberg found that the size and weight of the particles determined their rate of settling out, or sedimentation, and he used this fact to measure their size. With an ultracentrifuge, Svedberg went on to determine precisely the molecular weights of highly complex proteins such as haemoglobin. In later years he made studies in nuclear chemistry, contributed to the improvement of the cyclotron, and helped his student Arne Tiselius in the development of the use of electrophoresis to separate and analyse proteins.

Centrifuge proteins at high speeds in a sucrose gradient. Different part of ribosome will settle out at different layers.

Question 5

How can molecular biology be used to study the microbial loop?

Answer 5

• Importance of microbes in the recycling of nutrients in the upper ocean - microbial loop.
• Find out which microbes they are by sequencing samples of seawater.
• Cyanobacteria - synecogogus
  
  • Fixes vast quantities of carbon and produces O2 at a large scale.

Question 6

Describe the microbes present in the nitrogen cycle within the ocean.

Answer 6

• Many micro-organisms in the oceans we haven’t studied / don’t know are there
• SUP05 - only been able to detect by its gene.
• Nitrogen fixation - microbes fix nitrogen gas.
• NifH - gene responsible for nitrogen fixation.
• We can detect nifH gene in eDNA to find out if and how much nitrogen fixation is going on.
• Bacteriorhodopsin - bacteria bound to chromophores detect and convert light in your eye. Proteins in the back of your eye are called OPSINS.
• Evolved from photoactive proteins in bacteria which evolved the ability to see before we did.

Question 7

Describe the structure of DNA.

Answer 7

• DNA is a double stranded helix
• Nucleotides linked by phospodiester bonds
• Two strands are bound together by weak hydrogen bonding
• One strand is complementary to the other
• A-T C-G
• Phosphate deoxyribose backbone
• Each strand = complimentary sequence

Question 8

How do you purify bacterial DNA?

Answer 8

• Break cells open with enzymes (Proteinase K) and detergents, which break the membrane
• The enzyme lysozyme is used to break the cell walls
• Precipitated with sodium acetate and ethanol
• Or bind it to beads (newer and faster)

Question 9

Describe the use of restriction enzymes

Answer 9

• First restriction enzyme found EcoR1
• Cuts DNA at specific sequences
• Cuts to reveal “sticky ends”
• Methylases also used to protect DNA from restriction
• Restriction enzymes do not cut their own DNA utilising another enzyme called methylases.

Question 10

Who & whne was the PCR reaction invented?

Answer 10

• Invented by Kary Mullis in 1983
• He received the Nobel Prize in Chemistry in 1993
• Technique has revolutionized the study of DNA

Question 11

Descibe the three main steps of the PCR reaction.

Answer 11

• As illustrated in the animated picture of PCR, three major steps are involved in a PCR. These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture. Each step – denatauration (alteration of structure), annealing (joining), and extension – takes place at a different temperature:

1. Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.
2. Annealing: At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded “template” (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.
3. Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.

• With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

Question 12

Why do primers need to be added to PCR?

Answer 12

• 5’ 3’ directionality o DNA molecules.
• Need primers as part of DNA molecules as polymerase will not bind to single stranded DNA
• Molecular photocopier

Question 13

What does large scale sequecing require?

Answer 13

• Large-scale sequencing requires DNA to be broken into fragments
• Cutting (with enzymes)
• Shearing (with mechanical forces)
• DNA is duplicated into a vector
• Individually sequence
• Assembled electronically
• Shotgun sequencing

Question 14

What does polymerase do?

Answer 14

• DNA polymerase can add free nucleotides
• DNA polymerase cannot begin a new chain on its own as it needs to build onto a piece of dsDNA
• So dsDNA is necessary for DNA synthesis

Question 15

What does ligase do?

Answer 15

Links togther DNA fragments.

Question 16

What is sanger sequencing?

Answer 16

• Sanger sequencing is a method of DNA sequencing first commercialized by Applied Biosystems, based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.[1][2] Developed by Frederick Sanger and colleagues in 1977, it was the most widely used sequencing method for approximately 40 years. More recently, higher volume Sanger sequencing has been supplanted by “Next-Gen” sequencing methods, especially for large-scale, automated genome analyses. However, the Sanger method remains in wide use, for smaller-scale projects, validation of Next-Gen results and for obtaining especially long contiguous DNA sequence reads (> 500 nucleotides).

Question 17

How does sanger sequencing work?

Answer 17

• Uses a DNA template, primer, polymerase, and fluorescent nucleotides * DNA sample fragments separated into four lanes
• One for each nucleotide (A, T, G, C)
• DNA bands are then visualized by UV light,
• Positions of the different bands used to read the DNA sequence
• For given template DNA, it’s like PCR except: Uses only a single primer and polymerase to make new ssDNA pieces. Includes regular nucleotides (A, C, G, T) for extension, but also includes dideoxy nucleotides.
  
  • Uses a single primer and polymerase to one new ssDNA piece. Dioxynucletides are incorporated at random there are lots of different lengths of DNA. A lazer reads the fluorescence and length of the DNA strand.

Question 18

What is dye sequencing?

Answer 18

• Four different label
• Each of the four nucleotide chains has a different dye
• Individual dyes fluoresce at unique wavelengths
• Vast majority of sequencing projects
  
  • easier
  • cheaper

Question 19

Whta is sequencing by ligation?

Answer 19

• Ligase identifies the nucleotide
  
  • instead of polymerase
  • doesn’t create a second strand
• Ligase joins probe sequences
  
  • produces a fluorescence.
• Based on the fluorescence one can infer the identity of the nucleotide

Question 20

2 base encoding

Answer 20

• Construct library of Probes
  
  • Small fragments representing two bases
    
    • Combination results in sixteen unique probes
    • Each fluoresces at a different wavelength
• Sequencing Reaction
  
  • 2-base encoding is based on sequencing by ligation
• Decoding Data
  
  • Remember each color indicates two bases
  • Need to know one of the bases in the sequence

Question 21

What are the two main appraches to sequencing protiens?

Answer 21

Mass spec and edman degration

Question 22

What is bioinformatics?

Answer 22

• DNA sequencing and protein sequencing are providing vast quantities of data.
• Bioinformatics is the science of understanding the biological meaning of this information.
• Uses applied mathematics, computer science, statistics
• Databases
  
  • Genbank – 30 years old, grows exponentially doubling every 18 months, 150 billlion base pairs.
  • Uniprot (Universal protein resource) European based, 40 million sequences
  • Glycome database – carbohydrate structures
  • wwPDB protein data bank – structural information

Question 23

Summary

Answer 23

Molecular biology provides information about the molecules of life, DNA and proteins n DNA structure, extraction and cloning n Sequencing of DNA and proteins n Bioinformatics n Summary

Question 24

grant paper - genomics

Answer 24

Cock et al., 2010

Genomics = ‘the study of the structure, function and diversity of genomes’

Genomes = ‘the collective term for all the genetic information contained in a particular organism’

• genomic approaches differ from biological approaches in their scale – extends analyses from a smaller number of genes to a complete genome
• marine organisms were poorly represented amongst early genomic models, but this situation has been rectified in recent years due to the reduced cost of DNA sequencing
• reduction in cost has also facilitated the creation of new fields, e.g. metagenomics
• ‘genomic approaches are now being applied to a diverse catalogue of questions in marine biology’
• > including exploiting the extensive phylogenetic diversity of marine organisms ‘to explore the evolution of developmental processes, characterising the marine ecosystems that play key roles in global geochemical cycles […], and understanding ecological interactions within important marine ecosystems’

Question 25

How much of the genome codes for genes?

Answer 25

3%

Question 26

When was the term genome first used?

Answer 26

* Term “genome” first used by German botanist Hans Winkler in 1920 * 1986 Thomas Roderick used “genomics” for the mapping sequencing and characterization of genomes

Question 27

What causes the variability of genome structure?

Answer 27

* Duplication events * Transposons * Microsatellites * Repetitive DNA's

Question 28

Grant paper - what does the size of the genome imply?

Answer 28

Lessons from sequencing Primrose & Twyman, 2006 * à genome sequencing increases understanding of basic biology, e.g. ‘the larger the bacterial genome, the greater the metabolic capabilities of the host organism [meaning that] the organism can be found in a greater number of habitats’

Question 29

what is functional genomics ?

Answer 29

* Once we know the sequence of genes, we want to know the function * The genome is the same in all cells of an individual, except for random mutations * However, in each cell, only a subset of the genes is expressed * The portion of the genome that is used in each cell correlates with the cell’s differentiated state

Question 30

WIDER READING: functional genomics

Answer 30

* Carvalho et al., 2010 * STUDY: highlights the significance of genomics and genetic principles in explaining the interactions among different biological levels of diversity * - genomic methods = powerful in revealing previously undetected taxonomic, genetic and functional diversity, e.g. the identification of new species & metabolic pathways * - the ability to target specific gene structure and function has led to the discovery of new metabolic pathways, thereby increasing our ability to understand and explore functional links among marine biota, ecological processes and novel marine products’ *

Question 31

What are expression microarrays?

Answer 31

A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarraysto measure the expressionlevels of large numbers of genes simultaneously or to genotype multiple regions of a genome. * Global expression analysis * RNA levels of every gene in the genome analysed in parallel * Compare with Northen blot * Microarrays contain more infomation by many orders of magnitude

Question 32

WIDER READING: expression microarrays

Answer 32

Wu et al., 2001 * - to determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nutrient cycling * - results indicated that glass-based microarray hybridisation has potential as a tool for revealing functional gene composition in natural microbial communities * BUT more work is needed to improve sensitivity

Question 33

What are comarative genomics?

Answer 33

* **What are the adaptive traits of an organism?** * Mechanism of evolution * what is conserved between speies? * Genes for basic processes * What makes closet related species different? * Their adaptive traits.

Question 34

WIDER READING: Comparartive genomics.

Answer 34

Primrose & Twyman, 2006 * - comparative genomics is possible due to vast quantity of genomes that have been sequenced * - enables the comparison of distantly related species to decipher major stages in evolution * - comparing closely related species allows more recent events to be uncovered, e.g. genome rearrangement that has facilitated speciation * - analysis of bacterial genomes = most progressive area * - example of findings from comparative genomics: * ‘Comparative analysis of the genomes of a wide range of thermophiles has revealed [...] strong evidence of extensive horizontal gene transfer.’ [but the basis for thermophilic trait is yet to be discovered]

Question 35

Identofying adaptive traits

Answer 35

* Find functional bits of the genome by studying the compounds and which segments are responsible for traits - then isolate & copy. * Two watches - difference in parts is likely to relate to a difference in function.

Question 36

what are the application of comparative genomics?

Answer 36

* Disease * Diagnosis * Pharmacogenomics * Design therapies based on the individual’s genome * Subtle, but important, differences in genomes * Cause differences in how one responds to drugs * Identify those who will suffer harmful side effects from particular drugs

Question 37

WIDER READING: Diagnosis in disease - cancer

Answer 37

Groupings of gene expression patterns show clear * Jimeno et al (2006) run a marine anticancer program that uses the sea to develop new agents for resistant solid tumours * \> this review also described the integration of different pharmacogenomic tools in the development of marine-derived compounds currently in development * \> results ‘open the gates’ for customised therapies in cancer patients in the near future

Question 38

WIDER READING: Disease diagnosis from genomics - pharmacogenetics

Answer 38

Hedgecoe & Martin, 2003 * PAPER: analysis of the development of pharmacogenetics & its social and ethical issues * ‘following the genome project and the development of powerful new gene sequencing and analysis technologies, a new form of research known as association genetics has started to transform the study of the relationship between genes and disease’ = functional genomics * -\> this has enabled the understanding of diseases at the genetic level and the identification of disease-associated genes * - DNA arrays allow cheap, fast & large-scale screening of populations for genetic markers that are correlated to positive or negative reactions to specific drugs

Question 39

WIDER READING: ethical issues - genomics

Answer 39

**Cambon-Thomsen et al., 2007** * The main ethical issues with human biobanks (= ‘biological sample collections with associated personal and clinical data’): * - informed consent - confidentiality - secondary use of samples and data **De Vries et al. 2011** * - genomics research raises ethical and governance issues about sample export and ownership & the use of archived samples * - many ethical issues are raised when genomics research is conducted on populations in developing countries, e.g. populations included in MalariaGEN

Question 40

Genomics summary

Answer 40

* Genomics is the study of organism genome information * Genome mapping allows all coding regions to be physically mapped on the chromosome * Comparative Genomics is a powerful way to discover new biological information * Ethical and Legal aspects need to be considered

Question 41

WIDER READING : shotgun sequencing

Answer 41

**Primrose & Twyman, 2006** _Shotgun sequencing_ = genomic sequencing strategy whereby ‘large numbers of genomic fragments are sequenced and sophisticated bioinformatics algorithms used’ to construct the sequence à first success was the complete sequence of the bacterium H. influenzae (Fleischmass et al., 1995) à important to note that the merits of shotgun sequencing vs. ordered, map-based sequencing are still being debated

Question 42

What is proteomics?

Answer 42

* Proteomics is the large scale study of proteins including their structure and function * It is from the words ‘protein’ and ‘genome’ * The link between an organisms DNA and how it responds to its environment * Proteome – protein & genome * Looking at protein genomics of 100,000's of proteins. * Understanding an organisms full complement of proteins (protein enzymes and hormones – effecting an organisms behavior – very mechanistic understanding of an organism.

Question 43

In marine sciences where can proteomics be applied to?

Answer 43

* In marine sciences being applied to * Biotechnology * Environmental toxicology * Aquaculture * Natural products chemistry

Question 44

What is natural products chemistry?

Answer 44

Natural products chemistry – a low molecular weight product which has a particular biological or metabolic activity. (small chemicals with drug action e.g. penicillin is a natural product in this regard)

Question 45

State the processes for sequencing a peptide.

Answer 45

Gene expressed – (transcription) - mRNA produced - (translation) is the conversion of a mRNA molecule into a protein. Ribosome receives mRNA molecule an uses that sequence to convert it to an amino acid sequence - protein or peptide.

Question 46

What is Transcriptomics?

Answer 46

Transcriptomics - study of mRNA molecules in an organism.

Question 47

Protien protien interatcions.

Answer 47

* Within the field of proteomics we have protein – protein interactions – how does one protein effect the behavior of another protein. * We can also have interactions between protein and DNA. * DNA polymerase – makes DNA – protein - DNA interaction (area of biochemistry which is important and interesting. * Talking about a protein DNA interaction in an exam – talk about DNA polymerase. How useful the polymerase chain reaction was.

Question 48

What processes does collectig additional infomation - not just protien sequence require?

Answer 48

* Requires protein purification – not simple * Mass spectrometry * Post translational modifications * Phosphorylation – changes function - most well studied modification * Glycosylation – addition of carbohydrates to proteins eg glycans important for example in cell adhesion * Not simple because tissues will contain 1000's of proteins. * Purify ribosome – previous lecture * Mass spectrometry of proteins, if you know the weight of a protein you can match it against a known database. * Post translational modification * Protein can have a phosphate added – phosphorylation.

Question 49

What basic steps may a study into proteomics follow?

Answer 49

* Study of the important proteins in a marine sponge. * 1st step is often experimental design – how is the experiment going to be run. * Liquid chromatography or mass spec / mass spec * In silica analysis – inside a computer – looking in databases – identify proteins ect

Question 50

What two types of approches can proteomics take?

Answer 50

**Bottom up approach:** take all proteins in a particular organisms – break up into little peptides. Analyse structure and sequence of little peptides, interrogate database and reassemble the full structure of the proteins that are present. **TOP-down** - understand what proteins are present, separate them carefully and analyse them one by one with chromatography or mass spectrometry.

Question 51

What does genome annotation consist of?

Answer 51

* Genome annotation consists of adding biological information to known sequences or genomes * Most expensive part * ID non coding regions * Predict gene presence * Predict protein sequence * Predict function * Use homologies (BLAST) Allows the large amounts of non-coding DNA - junk DNA to be identified. If you can predict gene presence you can also predict protein sequence, and then you can often predict a function. Use terminology. Hypothesised proteins that you think should be there but can't find them / series of enzyme could suggest it can digest something which can be tested. One of the most common methods is using a blast search.

Question 52

What is a blast search?

Answer 52

Basic Local Alignment Search Tool * Enter a DNA sequence into the box, search, find DNA which matches your sequence. * Identify genes of interest. * You can therefore identify the genes of interest and the protiens of interest using blast searches

Question 53

Blast searches - flow sequence

Answer 53

* Flow sequence of work which involves you loading your particular target sequence into the database, predict proteins which are occurring. * BLAST N a particular type of search sequence which involves searching nucleotide sequences, NT nucleotide sequence database. * If you blast search in a protein database you will search with amino acid sequences.

Question 54

Describe mass specctrometry

Answer 54

Take a sample, ionise it by accelerating it with a laser, then propel the sample through a tube and pass it through an electromagnetic field. Because they are ionised they will fly through the electromagnetic tube they will be deflected. The deflection will change and the fragments which are the right weight to hit the detector will be recorded.

Question 55

WIDER READING: Mass spectrometry

Answer 55

Mass spectrometry Aebersold & Mann, 2003 - recent successes illustrate the role of mass spectrometry-based proteomics as a tool for molecular and cellular biology - includes the study of protein-protein interactions, the mapping of numerous organelles & the generation of quantitative protein profiles from diverse species - the ability of mass spectrometry to identify & precisely quantify proteins from complex samples is expected to impact biology & medicine

Question 56

MALDI TOF Matrix?

Answer 56

* MALDI TOF Matrix assisted laser desorption deionisation Time of Flight Mass Spectroscopy. * MALDI - means you zap proteins with a laser when they are stuck to a surface, they desorb which means they are blasted of the surface and they ionise by the addition of protons. Take the peptides and use a mass spec - good way to identify mixtures of protein.

Question 57

ITRAQ?

Answer 57

* Isobaric tag for relative and absolute quantitation: iTRAQ * Isobaric labelling method for quantitative proteomics * Allows study of numerous proteins in a single sample * Uses isotope labels * Two reagent types used 4 plex and 8 plex * Signals of the reporter ions allow calculation of the relative abundance of identified peptides * Signals processed using standard software

Question 58

WIDER READING: ITAQ

Answer 58

**iTRAQ** **Evans et al., 2006** = profiles the secretome of marine bacterium by using amine-specific isobaric tagging (iTRAQ) - proteins that are secreted from bacterium were labelled using iTRAQ, and identified using two-dimnsional SCX and nano liquid-chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) - iTRAQ labelling experiments enabled accurate measurement of the proteins identified - also obtained a sequence-base prediction of Pseudoalteromonas tunicata secretome - comparing the sequence with the expressed proteome suggests that this secretion pathway has a role in iron transport and acquisition

Question 59

WIDER READING: fish gonads ITAQ

Answer 59

**Fong et al., 2014** – looking at the response of exposure of fish gonads to a particular toxin BDE-47 = pollutant - proteomic analysis of those expressed inside the gonads using iTRAQ - can understand basic biochemical processes occurring inside the fish - investigated the protein expression profiles of male and female gonads of Oryzias melastigma exposed to dietary BDE-47 at 2 dosages for 21 days à extracted proteins were labelled with iTRAQ and analysed on a MALDI TOF/TOF analyser - among the 42 differentially expressed proteins in testis, down-regulation of histone variants implicated that BDE-47 may disrupt spermatogenesis and induce sterility in fishes - reproductive impairment was also shown from 38 differentially expressed proteins in ovaries

Question 60

Summary proteomics

Answer 60

* Proteomics is the study of proteins from a genome * Being widely applied in marine sciences * Techniques used include iTRAQ quantification * There is a requirement for annotated genomes * Growth area in molecular biology

Question 61

What is barcoding?

Answer 61

Barcoding * Amplified and sequenced regions of DNA * Need to be conserved and long * International consortium established * CBOL Consortium for the barcode of life * ibiol.org * Focus is on the 16s ribosomal gene

Question 62

Describe eDNA and Metabarcoding

Answer 62

Advances in molecular biology include High Throughput Sequencing allowing DNA from a range of environmental samples such as water or sediment (eDNA) to be processed in bulk at an increased rate. A range of genes and matching primers are used to compare species, mitochondrial genes such as cytochrome oxidase I (COI) are common target genes to compare species in the animal kingdom (Clarke et al, 2017). Sequences of eDNA can then be associated to known databases to give a taxonomic identification, in a process called metabarcoding (Creer et al, 2016).

Question 63

What is the Cytochrome oxidase 1 gene?

Answer 63

* COI gene a good example * Separates most species easily * Doesn’t work well with Cnidaria as they have very good mitochondrial DNA repair mechanisms in their mitochondria * COI also has low resolution in plant species so chloroplast genes are also being looked at * COI separate most species quite easily - variable * Cnidaria - COI gene does not mutate as fast.

Question 64

What is a fish barcoding database?

Answer 64

* FISHBOL.org = fish barcoding database * Assemby of all fish species in the world (freely available) * Advantage of these databases is that even small samples (fragments of fin, egg, larvae) can be identified * Predator prey studies * Larval identification (often tricky)

Question 65

Census of Marine Life

Answer 65

* Major international study * Diversity and distribution of marine organisms * 14 field projects * Barcoding initiative within COML * Complements but does not replace conventional morphological approaches Rapid sequence information gathering is a challenge

Question 66

Describe flow cytometry

Answer 66

Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.[1][2][3][4] A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam and the light scattered is characteristic to the cells and their components. Cells are often labelled with fluorescent markers so that light is first absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer. (rest in notes)

Question 67

Give another example like flow cytometry of sorting cells.

Answer 67

Another example involving the use of a high temperature process, which denatures DNA in particular cells. You then bind a fluorescent probe which binds to specific cells, that might have a particular sequence (COI for example). You can fluorescently labels cells in a species specific manner, then you can use flow cytometry to purify out particular subpopulation of cells.

Question 68

Give some applications of applying molecular biology (Flow cytometry) in the marine enviroment.

Answer 68

* Harmful algal blooms * Characterisation of complex multispecies communities * Fecal pellet analysis * Monitoring biodiversity hotspots * Distribution of fish larvae * Monitoring of pollution reduction by mangrove swamp communities * Real world analysis of a faecal pellet. Flow cytometry is used to study pop of microbes inside faecal pellets - pulling techniques together.

Question 69

What ways can you detect individual species?

Answer 69

* Species specific primers * Labelled with fluorescent dyes for detection (not sequencing) * RAPD – random amplified polymorphic DNA fingerprinting * PCR with two short random primers, lots of PCR products occur and visualised by gel electrophoresis * RAPD * Carry out a PCR reaction * Use 2 short random primers * Give a series of fragments and balancing pattern - barcoded

Question 70

How can nanotechnilogy be used in molecular biology?

Answer 70

* Quantum dots – 2-10 nm crystals emit light of specific wavelength after laser excitation * Can use to detect specific DNA sequences * QD coupled to streptavidin (high affinity to biotin) * DNA probe plus biotin connect the laser light to detection of DNA sequence in a microcapillary

Question 71

How do quantum dots work?

Answer 71

DNA-functionalization of quantum dots is the attachment of strands of DNA to the surface of a quantum dot. Although quantum dots with Cd have some cytotoxic release, researchers have functionalized quantum dots for biocompatibility and bound them to DNA in order to combine the advantages of both materials. Quantum dots are commonly used for imaging biological systems in vitro and in vivo in animal studies due to their excellent optical properties when excited by light, while DNA has numerous bioengineering applications, including: genetic engineering, self-assembling nanostructures, protein binding, and biomarkers. The ability to visualize the chemical and biological processes of DNA allows feedback to optimize and learn about these small scale behaviours.

Question 72

What are microarrays?

Answer 72

* Glass slides * Hundreds of thousands of spots of oligo probes specific to certain genes Generally used to study changes in gene expression * Also used for detecting species * Community arrays * Sample amplified – amplified product is labelled * If there is hybridisation of the labelled DNA to the probe this indicates a certain species of gene is present * Data spots - genes not expressed in that particular tissue of that fish.

Question 73

Summary using molecular biology

Answer 73

Molecular tools are revolutionising how we view marine biological diversity Barcoding Flow cytometry with molecular biology RAPD fingerprinting Quantum dots Microarrays

Question 74

Where are lots of methane hydrates found?

Answer 74

Lattice of methane. Lots of frozen marine clathrates in marine sediments, which may release methane.

Question 75

What produces methane as a way of degrading organic matter?

Answer 75

* Methanogens produce methane as a way of degrading organic matter. * Archaea which produce methane * (Archaea –Woese 1977 three domains of living organisms based on 16SrRNA sequences) * Responsible for methane production in sediments and animal gut * Can be tracked and detected using molecular techniques

Question 76

Whta is chemolithotrophy?

Answer 76

* Rock eaters * Low oxygen favours chemolithotrophy * N loss * Production of climate active trace gases nitrous oxide (N2O) and methane (CH4) * Carbon cycle (ability of phytoplankton to uptake carbon dioxide) affected by OMZ’s

Question 77

metagenome

Answer 77

Total genome of a particular sample

Question 78

Summary - what can molecular techniques provide information on, about nutrient cycling?

Answer 78

* Nutrient cycling * Molecular techniques can provide information about the presence and distribution of important nutrient cycling organisms * Monoclonal antibodies as well as DNA based methods can be used to provide information about trophic linkages in marine ecosystems

Question 79

List all the molecular methods available.

Answer 79

1. DNA sequencing 1. pyrosequencing 2. Genomics 3. Proteomics 4. Metabolomics

Question 80

What is pyrosequencing?

Answer 80

* Pyrosequencing is a method of DNA sequencing that differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release and the generation of light on nucleotide incorporation, rather than chain termination with dideoxynucleotides. Pyrosequencing methods for identification of RGM rely on a 20–30-bp region within the hypervariable region A of the 16S rRNA gene. * Pyrosequencing is another rapid DNA sequencing technology based on sequencing by synthesis * Relies on the detection of pyrophosphate release upon nucleotide incorporation * (compare with Sanger method for example which relies on chain termination) Nucleotides added one at a time and when the “right one” is added and incorporated a light emitting reaction occurs

Question 81

Why bother making cDNA libraries?

Answer 81

* Eukaryotic tissue is very difficult to handle in the lab. The easiest thing to do in the lab is to convert the transcript into DNA (can be any tissue) * Lyse the cells, and purify out the mRNA. * Can do fairly clever manipulations in the test tube, which help you to create a small double stranded piece of DNA at one end, by adding a poly - T primer. Then we can actually carry out production of DNA by reverse transcriptase (literally reverse transcription). * EXAM question - why bother making cDNA libraries - because it is much easier to handle than sDNA.

Question 82

Whta is an EST analysis?

Answer 82

* Expressed Sequence Tag is a short sequence from a CDNA sequence * Can be used as a short simple label to identify genetranscripts * Saves having to sequence / analyse the whole gene * A short sequence from a cDNA sequence. If you carry out with a eukaryotic or genomic gene, you can see that you have a gene from a eukaryote. Genes are often made up of exons, when you have cDNA you will have small pieces with match exxons. If we have cDNA pieces, when we put them together to make a single cDNA we can create primers which are EST (expressed sequence tag) which can be used as a short and simple label which identifies, using PCR, the presence of a particular gene transcript. Where we have test tubes full of cDNA molecules from organisms, you can find out what is in there by using PCR. Specific EST will say if a transcript is present in that tissue or test tube.

Question 83

What is KEGG?

Answer 83

* Kyoto Encyclopaedia of Genes and Genomes (KEGG) * KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies

Question 84

Summay of molecular methods.

Answer 84

Summary of molecular methods n Pyrosequencing technology n Example – Rainbow trout and Gene Ontogeny n KEGG analysis in metabolomics n Molecular biology methods are powerful tools in understanding and analysing biological processes of relevance to aquaculture and fisheries