Using Molecular Biology Flashcards
What are the uses from Molecular Biological tools?
- 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
What did Carl Woese develop as a way to study evolutionary relationships of bacteria?
- 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.
What is a ribosome made up of?
- 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
Who was awarded the nobel proze for for his research on colloids and proteins using the ultracentrifuge, and what did they do?
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.
How can molecular biology be used to study the microbial loop?
- 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.
Describe the microbes present in the nitrogen cycle within the ocean.
- 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.
Describe the structure of DNA.
- 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
How do you purify bacterial DNA?
- 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)
Describe the use of restriction enzymes
- 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.
Who & whne was the PCR reaction invented?
- Invented by Kary Mullis in 1983
- He received the Nobel Prize in Chemistry in 1993
- Technique has revolutionized the study of DNA
Descibe the three main steps of the PCR reaction.
- 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:
- Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.
- 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.
- 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.
Why do primers need to be added to PCR?
- 5’ 3’ directionality o DNA molecules.
- Need primers as part of DNA molecules as polymerase will not bind to single stranded DNA
- Molecular photocopier
What does large scale sequecing require?
- 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
What does polymerase do?
- 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
What does ligase do?
Links togther DNA fragments.
What is sanger sequencing?
- 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).
How does sanger sequencing work?
- 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.
What is dye sequencing?
- 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
Whta is sequencing by ligation?
- 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
2 base encoding
- Construct library of Probes
- Small fragments representing two bases
- Combination results in sixteen unique probes
- Each fluoresces at a different wavelength
- Small fragments representing two bases
- 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
What are the two main appraches to sequencing protiens?
Mass spec and edman degration
What is bioinformatics?
- 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
Summary
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
grant paper - genomics
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’
How much of the genome codes for genes?
3%
When was the term genome first used?
- Term “genome” first used by German botanist Hans Winkler in 1920
- 1986 Thomas Roderick used “genomics” for the mapping sequencing and characterization of genomes
What causes the variability of genome structure?
- Duplication events
- Transposons
- Microsatellites
- Repetitive DNA’s
Grant paper - what does the size of the genome imply?
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’
what is functional genomics ?
- 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
WIDER READING: functional genomics
- 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’
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- 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’
What are expression microarrays?
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
WIDER READING: expression microarrays
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
What are comarative genomics?
- 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.
