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Molecular Biology Exam Cheatsheet
Bachelor of Forensic Science (University of Technology Sydney)
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Nucleic Acids & Electrophoresis
Denature by breakage of H-bonds by heat.
Alcohol less polar than water > decrease dielectric dependence on DNA conc. Transformed after
contact with water, and high voltage. Creating
o read sequences from DNA gel go from bottom Guanidinium isothiocynate > denatures prevents constant of water/alcohol mixture > less able to
up… 5’ at bottom, 3’ at top.Maybe other around
H-bonding reforming in RNA.
hydrate/solvate DNA. Plasmids semi-purified from transient pores and moving the outside plasmid
inside.
around if A is first?
Structure etc. of chromosomal DNA
bacterial cell lysates using detergent Restriction
Transformation Effic. = [#transformants/ug of
DNA is bases, sugar, phosphate. Base-pairing and DNA strand open apart by enzyme (helicase) then Endo & Ligation
read by RNA polymerase into mRNA transcript. Blunt ends always compatible, staggered require plasmid DNA] max no. of transfor. = 108/ug DNA.
complementary strands.
LacZ-B gal complement: gene of interest,
compatible ends.
H-bonds between nucleotide bases (non-covalent) Supercoiling > gyrases/topoisomerases effect
hold two strands together. Outside is polar due to conversion in either direction – DNA supercoiled Restict. Enzymes act as protectors against foreign inserted on Lacz gene. Now LacZ not function (no
lactose used)/ X-gal (sugar) turns blue of bacteria
in resting state, relaxed when genes transcribed
invasive DNA. Cut DNA palindromes (reverse
sugar/phos.
sequence of two strands, same sequence direction), can break down to lactose (no transformaition)
H-bonds between horizontally stacked base pairs etc.
White = colonies of interest with broken LacZ
Gentle lysis to minimise breakages, detergent and some cut same site (isoschizomers – cut same
in core of molecule; hydrophobic interactions
high salt added to remove unwanted by
between vertically stacked base rings in core;
palindrome as same sit; digestions generate new gene, cannot digest x-gal. No growth = cells with
phenol/chrlogorm extraction.
5’P and 3’OH ends. Specfic reaction requirements no plasmid uptake as they were killed by amp.
sodium neutralises oxygen negative charges on
SDS has negative chargers that dissociate the
of puffer pH, low, medium/high ionic strength and Blue = vector relegation = efficient with no
acidic phosphate on outside of molecules.
electrostatic and polar interactions on the protein cation and reaction temp. Fastdigest enzyme used plasmid uptake as killed by amp.
Bases are non-polar/alkaline pyramidine 6 ring
surface. SDS non-polar lipid chains can invade and as universal buffer and can complatr unit reaction. Lower white colonies = recombiant plasmids
C,U,T: Purine double ring A,G.
transform less efficiently.
unravel the protein non-polar core.
Neoschizomers > enzymes that recognise same
DNA is negatively charged and migrates from
Directional/forced cloning = solving problem of
DNA clump together when salt-alcohol precip.
equence by cuts at different positions.
cathode to anode. Podsitions of discrete DNA
Salt neutralises charge and alcohol reduces
Bacterial restriction-methylation system is part of vecto relegation. Reducing background to -.
bands can be locate by staining, visualised at
Double digest. EcoR1&BamHI cannt re-ligate.
polarity
254/302nm. Absorbed light shows 590nm.
bacterial host and protectivre integ of bacterial
Lots of streaking is too much shearing force.
genome. Screens invasive foreign DNA and will Alkaline phos removes 5’ phosphates, no vector
Agarose conc. > efficient range of separation
relegation
Chromosomal DNA is streaky.
be restricted into many small pieces so DNA
depends on amount of agarose.
cannot recombine. Adds bulky methyl groups to A white colonies showing that inserting has
Conformation of DNA > supercoiled, relaxed and Abberations occur from irregular chromoDNA
inserts, mutations, deletion. Becomes useless.
linear of same molecular size migrate through
to C to every recognition site. Protected from self- interrupted lacZ region in the vector and
Amino acids can be specified by two/more codons digestion as methyl group prevents enzyme from complementation with the bacterial host LacZ is
agarose at different rates.
not possible.
= DNA and mRNA can be less specific and less
binding across site.
O more electronegative than H, having dipole
Modifying restric. Sites. Linkers (connect blunt),
conserved = will tolerate single base substitions. DNA transformation > sent to production
effect. Solvation is when dissolving compounds
adaptors (blunt + read made sticky), methyl,
with chargers or polar groups in water.
Diff codons for same amino acid may be used in Molecular cloning = copy paste
polylinker MCS recognises multiple.
Alcohols, methanol, ethanol etc. have terminal OH diff. parts of polypeptide sequence.
DNA ligase is the enzyme that joins DNA ends
groups and lone paired are able to engage in HTheoretical yield = vol. cell culture (ml) x cell
together. Catalysed by T4 DNA ligase that works Genetic Diseases & Lac Opron
bonding in water, which is why they are soluble
density (cells/ml) x chr. Size (bp x MW (dal/bp) x as non covalent dimer which monomer binding
Operon = 2+ genes expressed from single
and have high boiling points. . Butanol is long
AMU (g) Plasmids
across gaps in DNA strands. Esterification reaction promotor.
aliphatic chain that dominates it’s properties so it Elements that exist outside chromos. Replicate
new phos bonds to site and formed on each strand Genetic mutations spontaneous, chemicals.
is much less soluble. Isopropanol has a lower
with chromosome (stringent) and inherited by all with elimination of -OH and H. Requires ATP and Transition = purine/”” or pyrimidine / “” swap
dieletric constant than ethanol, making the
Transversion = purine/pyrim swap. Frameshift:
daughter cells, may be transferred by conjugation Mg.
water/iso mix less polar than an ethanol/water mix, or trandofmrtion into bacterial cells as naked
Staggered ends anneal more easily than blunt
deletions/insertions > translation shift in triplet
allowing nucleic acids to aggregate more readily. DNA.
when ends collade at low temp, they stick together codons sequence out of frame.
by complementary h-bonds, and DNA ligase binds Possible outcomes depending on carbon sources o
Water/hydrogen + RNA
Transformation process with CaCL2 …
Ribosomal (rRNA) > two long RNA molecules
Isolating plasmids requires selective removal of all to site and joins in new phos bonds covalent. More When glucose is oresent and lactose is absent
that associate with proteins to form two composite other macro components (polysacch, chromosomal controlled and less enzyme required. Blunt ends E.coli does not produce B-galactosidase o When
ribosome units and third smaller rna (28s, 18s, 5.8s DNA, RNA, proteins, lipids, membrane fragments. don’t stick, efficient at low temp slows movement glucose is present and lactose is present E.coli
of DNA with corowding, achieve by adding
E; 23s, 16s, 5s P. Helps form assemblies with
DNA in high salt buffers binds to hydroxylated
does produce D-alactosidase, but at very low level
soluble polymer to mix. Does not anneal and needs o When glucose is absent and lactose is absent
attached protein/enzyme. 80% cellcular RNA.
silica discs by displacing water, sub NA+ DNA
more enzyme.
mRNA transcribed from specific gene sequence. interactions with silica hydroxyl groups, DNA
E.coli does not produce Bgalactosidase o When
Used as templates to synthesis complimentary
eluted from disc when high salt replaced by pure DNA Transformation. Mol cloning strategy.
glucose is absent and lactose is present E.coli does
DNA.
water, removing sodium, releasing DNA and regen DNA must be competent for trans in ice CaCl2
produce Bgalactosidase, at a very high level
RNA is susceptible to degradation
strong H-bond interaction of waater.Silica-salt acts Ectroporation of DNA > circular DNA electro
Tangier Disease: ABCA1 cholesterol transporter
as a bridge between and separates…
competence by washing in cold water, size
impaired: ^ LDL, low HDL à Atherosclerosis,
dependence has much upper limit. Linear uptake cardiovascular disease. CF:CFTR protein
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impaired: no Cl- out of cell, no water flow à ^
polymerase, 4 types of dNTP but only one type of evolutionary factor between pro/eukaryotes will
One thing that can be odd about DNA linear
mucous. Muscular Dystrophy: X-linked,
ddNTP(dideoxynucleotide triphosphate) =
stop transcription. Seen when multiple triplets
standards are DOUBLETS.
Dystrophin mutation = shock absorber in muscles. terminator, lacks 3- hydroxylOH group at 3’
specify same AA. Solution: e.coli used in hosting Ligation from blunt cuts makes new sites different
carbon). Termination = different length DNA
plasmid with copies of the genes for rare tRNAs. to either of the original sites and unable to be cut
Colour Blindness: x-linked, recessive • Lac
4) Protein Folding + stability: foreign proteins
Operon: Lactose (sugar in milk, genes control its fragments. Can only be done for DNA strands
with either enzyme. STICKY??
between 100- 1,000bp.
subject to misfolding. Solution: use less proteases, Add the plasmids in each lane to determine if they
usage), cells prefer glucose. No Lactose= Lac
DNA Sequencing Electrophoresis: read from
repressor binds to operator, no transcription as
protease inhibitors, foldase protein, chaperone.
match the 1st. This will determine if it’s the same
bottom (5’) up to (3’). Polyacrylamide Gel used: 5)Fusion/ hybrid: joining NàC terminus of part of plasmid or different.
RNA polymerase cannot bind. Lactose, No
more dense, ^voltage to separate DNA strands by 1 one protein to another. Used for detection of
Glucose = Allolactose binds to repressor, which
When restriction sites provided count between
promoter-less gene. Proteins created through
leaves operator site, transcription occurs. CAP = base pair. • Cycle Sequencing: each ddNTP is
each one!
labelled with different fluor tag & all 4 run in lane fusion of genes to more stable in e.coli, 3 vectors The only thing different about an insertion is that it
catabolite activator, cAMP + Protein = bind to
of gel (unlike standard methods). Same tube. Gel give possible frames for hybrid structure.
CAP site ^transcription, (low glucose, ^cAMP).
is ligated in one direction ONLY and in the Smalcan be shorter (15-20cm). • “Shot-gun’
Frameshift will alter sequence if not in frame/
Glucose + Lactose= Allolactose binds to Lac
EcoRi site.
repressor, no repressor binding, transcription, Low Sequencing: for long DNA strands (>1Kbp), build premature stopcodon. • His-Tag = peptide tag, only Deduce that crowding due to the presence of the
up an overlapping map of shorter sequences. •
cAMP = ^glucose, CAP site not activated: less
proteins with tag will stick to nickel column.
polymer IMPROVES the yield of blunt and
Bioinformatics: using computer technology to
Affinity purification for the expressed hybrid
transcription PCR
staggered end ligations. Undercrowding?
analyse DNA sequences. (relatedness of twins with protein. His tag à Cterminal.
Sample>DNA>PCR (thermal cycler)>DNA>
Mixing equal molar ratios of each set of molecules
parents) • RFLPs: (restriction fragment length
Amplification>Electrophoresis
To estimate the size of the plasmid use linear
can maximise the number of recombinant
Reaction Mix: DNA template, dNTP mix, buffer, polymorphisms) used to detect genetic
conformation (not relaxed/supercoiled)
plasmids.
Mg, primer pair, water, Taq polymerase Optimal abnormalities. WT & Mutants have different
It is true that variation in widths is due to diffusion Using rectriction enzymes that generate blunt ends
fragment sizes. Mutant has base change directly
temp: 72-78*C, not reactive at RT. Reverse
of wider bands, size of DNA may be estimated,
rather than staggered ends is because using
opposite same as WT – so target codon changed
transcriptase (mRNA>DNA)
bands bind an amount of Gel-Red that is
staggered ends require compatible enzyme ends,
into new AA. • Tm= 2(A+T) +4(G+C). 3 H-bonds proportional to size of fragments and difficult to limiting enzyme choices, whereas blunt ends are
Thermal Cycler: 1) denature 95’C 2) Anneling
primers 56’C 3) Extension 72’C. 1kb of DNA/min, between CG, more CG in sequence = ^Tm •
estimate size due to WIDTH of bands, length is
always compatible.
30 cycles = ½ billion DNA copies. Many cycles- Polymorphisms: discontinuous genetic variation à easier. It is incorrect that DNA bands reflect the
When there is an overhang, reacting it with certain
different forms of individuals among the members amounts of DNA present and that it determines
^number of mutant plasmids Theoretical Yield:
things will be cut back to the prime end. If it is the
of a single species Vectors and Protein Expression width/staining intensity.
[mass(ng) x 2#cycles]=_ng. /109 = _g Actual
other way around, it will be added.
Vector Expression: controlled at gene (strong
Extension time: based on enzyme.
The smaller the mass of each inserted, the less it Linkers have extra bases either side of palindromic
regulated promoter), mRNA & protein level.
Primers: specific to DNA sequence. Amplified
can be seen because the fragments are too few and sites because endonucleases can’t cut, just to
product is restricted to primer sequence. Tm: 66- Features: RBS, transcription terminating sequence, short, however, they are still stained.
reassure.
Antibiotic resistance, Ori, repressor(protein that
70*C. Ideal Primer= lots of CG at 3’ end. Not
When a chromosome is transcribed into mRNA, it Silent = no change, vas and leu both have small
good = long CG regions (intermolecular bonding). bind to operator) gene & promoter-operator region. is done via the non-coding-template strand.
non-polar side chains, almost identical can be
If foreign DNA is inserted into say tetracycline
Primer Dimers: primer attaching to each other, 2 Promoter system regulated by heat sensitive
substuted with eachother. Missense is catastrophic.
repressor: 32’C (fully functional, no expression), area and not ampicillin. It will stay ampicillin
bands in gel instead of 1. Solution: 1) use less
Asardic is polar, acidic, can never exist inside
37’(partly functional, little expression), 42’C (not resistant and tetracycline susceptible.
primer 2) MgCl2 3)^Template DNA 4) Adjust
protein. Nonsense = protein stops if at the end
functional, max expression). • cDNA from
temp 5) Redesign primers
Stringent replication control…host natural plasmid though no big deal. Frameshift, entire shift of
RT-PCR: monitors DNA accumulation RT. SYBR mRNA:1)Viral transcriptase with poly AAAA tail, may be replicated and inherited by the dividing
code. Serine is polar so catastrophic with non2) Oligoid (dT) primer binds, 3) NaOh degrades bacterial cells.
green only binds to dsDNA. Fluor only emitted
polar.
mRNA, hairpin loop at 3’. 4) DNA pol. Extends Alkaline lysis is the best process…it removes
when attached to dsDNA.
If there are 80 exons, there are 79 introns
RAPD-PCR: Locating unknown sequence in any loop, 5) S1 Nuclease (ss specific cleaves hairpin chromosomes along with proteins and lipids in
Everything in operon requpred for lactose
loop end à 2 antiparallel dsDNA à amplification. • snow. Alkali denatures both with the chromosomal Structural genes only need one mRNA transcript,
genome using random primers.
Site-directed Mutagenisis: introducing mutations Factors Affecting Expression of Cloned Genes: 1) fragments, unable to renature and are precipitated, the regularoty on the left is separate!
by editing a pre-existing plasmid.
Promoters: strength of promoter depends on
circular plasmids will renature when neutralised. Wild-type/mutant primer pair bases to be
degree of consensus & distance between regions. Small plasmids survive.
substituted must be aligned precisely opposite one
DNA Sequencing, Bioinformat
Consensus = where DNA pol. binds: High (base
Binding with silica beads occurs due to the binding another because it ensures that the target codon
Reading frame: (Start ATGà stop codon)
changes) = less binding, Low (less base changes) = being assisted by Na+ that interacts with
will be changed to the new amino acid codon.
DNA Sequencing: get complete order of
^ binding. 2)RBS: in relation to +1 nucleotide of hydroxylated silica. The DNA release is affected Higher GC content = higher melting temp/degrees
nucleotides in DNA • 1) DNA amplification 2)
by the silica-sodium DNA interaction being
Vectors provide all possible frames for the hybrid
Heat-denatures ds to ssDNA 3)Primer anneals to 5’ mRNA transcript/ upstream promoter.
Transcription start point = 1+, 3)Codon Bias:
reversed by excess water.
construction.
end of DNA. 4) 4 different tubes (all have DNA
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Every copy has the mutation, it is always effective.
No transcription for low glucose and low lactose.
High glucose/lactose low level transcription does
not bind effectively. Highcamp/lact max
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