BIO2015: Lecture 2

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  • Created by: LMoney
  • Created on: 11-05-14 23:02
what is the most common approach to identifying presence of genes within the genome of an organism?
hybridization with radioactively labeled DNA or RNA probes- give both qualitative and quantitative information about the presence of the gene/genes
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double stranded nucleic acids can lose their secondary structure how?
by denaturation
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what is denaturation promoted by?
heat, extremes of pH, or hydrogen bond breaking agents such as concentrated urea solution
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Under similar conditions, temperature at which long DNA strand denatures depends on what?
base composition- DNA with higher (G+C) content denatures at higher temperature than DNA with lower (G+C) content, because the G-C base pairs are held together more strongly than A-T base pairs (3-hydrogen bonds in G-C bond vs 2 in A-T)
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what results in increase in absorbance of DNA?
strand separation
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what is hybridisation?
process by which separated DNA/RNA strands reform double helix
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when will strands hybridise?
if transferred to conditions favoring base pairing, cooling temperature to less than 70C favours double helix
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what other conditions are necessary for hybridisation?
High salt conc. also favours double helix formation as does removal of denaturants
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what kind of strands does hybridisation occur between?
complementary (or almost complementary) to each other i.e. in mixture of single strands- each strand singles out original partner to form double strand
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how long does hybridisation take?
Can take days if DNA is very dilute or contains very large number of different sequences
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what does the southern blotting technique detect?
specific DNA fragments, technique can be used for applications such as determining how many genes corresponding to particular cDNA probe are present in organism’s genome, many proteins are encoded by small gene families rather than single genes
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what do northern blots detect?
mRNA- gives size and amount of RNA
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what is the procedure for northern blotting?
1) separate RNA by agarose gel electrophoresis- separation by size 2) blot RNA onto nylon or nitrocellulose membrane 3) incubate membrane with labelled probe 4) locate hybridizing bands by autoradiography
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what are microarrays?
array of DNA molecules spotted onto glass
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what is the first of the 2 main types of microarray?
DNA (e.g. produced by PCR) is spotted onto slides
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what is the second?
2)Oligonucleotides are chemically synthesized attached to glass wafers- oligonucleotide sequences are based on known gene sequences
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what are microarrays good for?
look at changes in expression of large numbers of genes simultaneously, for analyzing differential gene expression
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how are microarrays used for analyzing differential gene expression?
1) 2 mRNA samples are copied to cDNA. Each cDNA population is labelled with a different fluorochrome • 2) mix cDNAs and hybridize to microarray • 3) fluorescence colour indicates whether one cDNA hybridizes
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where is PCR used?
in cloning, as probes, and in forensics. Also can be used to amplify rare specific DNA sequences from a complex mixture when the ends of the sequences are known and • PCR amplification of mutant alleles allows detection of human genetic diseases
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what are the requirements for PCR?
1) Taq DNA polymerase (copies original and generates new strands) 2) Oligonucleotide primers (forms initiation site for Taq polymerase) 3) • Mg2+ ions- stabilize primer:DNA duplex, required for correct dNTP incorporation
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what are required as the building blocks for the new DNA strands?
dNTPs (nucleotide bases that form the new DNA strands)
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how can the PCR reaction be optimised?
1)annealing temperature of the primers 2) concentration of Mg2+ in the reaction 3) extension time 4) denaturing and annealing times 5) extension temperature 6) amount of template and polymerase- more is less
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how can the annealing temperature?
1) primers have calculated annealing temperature (e.g. 54C) 2) temperature must be confirmed practically 3) temperature steps of 2C and above 4) use gradient cylinder
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how can Mg2+ conc be optimised?
fidelity of PCR depends on [Mg2+]: Vary [Mg2+] in steps of 0.5mM, sometimes a compromise must be made between yield and specificity
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in designing a PCR primer, how long should the primer be?
around 20 bases long
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what should the G/C content of a primer be?
45-55%
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how far should annealing temperatures be apart?
within 1 Celsius of each other
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what should the 3' most base be?
a G or C
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what should primers not do?
must not base pair with each other or form hairpins, and avoid repetitive DNA regions
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what happens when a primer forms a hairpin?
primer may be self-complementary and be able to fold into hairpin- 3’ end of the primer is base-paired, preventing it annealing to the target DNA
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what else could a primer form?
primer may form dimer with itself or with other the primer- primer dimers can be an excellent but unwanted substrate for the taq polymerase
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what is the most versatile tool (PC programme) for designing primers?
Primer3 at the whitehead institute
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how is DNA cloning accomplished with vectors?
DNA fragment of interest is placed into a vector DNA molecule which can replicate in a host cell. When single vector containing a single DNA fragment is introduced into a host cell, large numbers of the fragment are reproduced along with the vector
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what are 2 common vectors?
E.coli plasmid vectors and bacteriophage λ vectors
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what are plasmids?
extrachromosomal (i.e. separate from main genomic DNA of the organism) self-replicating DNA molecules- exist in multiple copies (a few 100’s) in bacterial cell
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what are plasmids function in nature?
to enhance adaption of bacteria to environment by transferring genes horizontally- i.e. without normal conjugation
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what has this ability of horizontal transfer resulted in?
responsible for antibiotic resistant pathogenic bacteria in hospitals etc. due to transfer of plasmids containing antibiotic resistance genes between bacterial spp.
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which other bacterial functions are carried on plasmids?
infective capacity (soil bacteria), toxin production, resistance to pollutants, etc.
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what are the 2 vital features of plasmids?
1) a replication origin, enabling the plasmid to be replicated independently of the bacterial 2) gene which confers selective advantage on organism (antibiotic resistance, toxin production, etc.) and selects for the plasmid
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what happens when no selective advantage is gained by the presence of a plasmid?
replication of a plasmid places a metabolic load on the host organism- thus plasmids tend to be spontaneously lost- normally present at low levels in microbial population
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what does the replication origin determine?
defines replication group of the plasmid- given replication origin may function in very limited, or wide range of spp. (narrow range or broad host range origins of replication), also determines plasmid copy number per cell
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what happens when fission of bacterial cells occurs?
plasmids are passed on to daughter cells
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how many plasmids of a given replication group can be stably maintained in bacterial cell simultaneously?
1- requires simultaneously selection for different characteristics carried on different plasmids (e.g. resistance to 2 different antbiotics)
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when is it best to purify plasmids?
after lysis
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purification relies on which differences in physical properties between plasmid DNA and genomic DNA?
1) different sedimentation in density gradient 2) different behavior on denaturation 3) ion exchange behaviour 4) adsorption behavior on surfaces
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what kind of molecule does purification result in?
circular ds DNA molecule- usually with added supercoil
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what are the yields in purification?
low- of the order of 1-100 micrograms per litre of culture, depending on plasmid copy number per cell
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how can plasmids be reintroduced into bacteria?
by transformation with naked circular ds plasmid DNA
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how are bacterial cells made ‘leaky?
by treatment with membrane permeabilising agents (e.g. calcium ions) these cells are called component. Component cells are mixed with DNA and subjected to mild heat shock (increases efficiency of transformation)
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how can DNA uptake be increased?
electroporation
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DNA is taken up at very low efficiency, how is this balanced?
plasmid then replicates in cell and daughters
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Efficiency of transformation falls rapidly as size of circular DNA increases or decreases?
increases- plasmids bigger than 10kb are difficult to transform
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when can cells which have taken up plasmid DNA can be isolated?
if a suitable selectable gene is present on the plasmid
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how else can bacteria transmit plasmids?
by conjugation- occurs at low frequency requires expression of correct transfer factors
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E. coli cells are engineered to synthesise which incomplete, inactive enzyme?
the w-peptide (omega peptide)
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what do cells containing original vector plasmid also synthesise?
a small peptide fragment from B-galactosidase- the a-peptide (alpha peptide)
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a-peptide binds to w-peptide to form what?
B-galactosidase
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do cells with original plasmid produce a blue or white colony when treated with X-Gal?
blue- in plasmids with an insert- gene is interrupted- alpha peptide is mutated and colonies are white
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what must be used to clone larger DNA fragments?
cosmids (and other vectors)
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what are BAC and YAC vectors?
bacterial artificial chromosome (up to 200kb) or yeast artificial chromosomes (up to10^6 kb)
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what is the basic structure of cosmids?
plasmid containing a cos site, so DNA can be packaged into phage particles in vitro
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what are the advantages to cosmids?
1) can carry larger DNA inserts than bacteriophage vectors because there are no bacteriophage genes 2) infect like viruses, replicate like plasmids
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what essential elements of a yeast chromosome do YACs have?
1) DNA replication origin (ORI) 2) Centromere (CEN) 3) Telomere (TEL)- Thus insert will replicate in yeast cells as part of artificial chromosome
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what is the maximum number of kbp that BACs can hold?
roughly 300kbp- (typically around 150kbp though) based on natural F factor plasmids
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how many copies of BACs per cell?
only 1 or 2
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how is transformation accomplished in BACs?
electroporation
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why in practice is production of DNA fragments greater than approx.. 40kb in length very difficult?
DNA is sensitive to physical shearing during isolation
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double stranded nucleic acids can lose their secondary structure how?

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by denaturation

Card 3

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what is denaturation promoted by?

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Card 4

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Under similar conditions, temperature at which long DNA strand denatures depends on what?

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Card 5

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what results in increase in absorbance of DNA?

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