Gene Tech
Gene Tech
DNA to RNA to Proteins
structure & function of DNA
DNA → deoxyribonucleic acid
1 nucleotide
1 5-carbon sugar (deoxyribose)
1 phosphate group
1 nitrogenous base
pyrimidines (one-carbon nitrogen ring)
thymine
cytosine
purines (two-carbon nitrogen ring)
adenine (≠ adenosine)
guanine
5’ to 3’ connection between nucleotides are phosphodiester bonds
phosphate group at 5’ and OH at 3’ get dehydrated
opposite strands of polynucleotides
form a double helix
strands must be antiparallel
one strand runs 5’ → 3’ while the other strand runs 3’ → 5’
adenine and thymine complement each other
2 hydrogen bonds
guanine and cytosine complement each other
3 hydrogen bonds
DNA stores genetic information on protein production
1 gene codes for 1 protein
DNA is able to
replicate itself
transmit information to offspring
directly synthesise proteins
mutate
genome → chromosome → DNA → genes
DNA replication
helicase
binds to DNA and forms replication bubble by unwinding and separating the 2 DNA strands
DNA polymerase
build new DNA strands that will pair with each old DNA strand via complementary base pairing
where there is an A on the old strand, polymerase adds a T to the new strand
semi-conservative model
half of the new DNA strand is made from the original DNA strand
enzymes use each strand as a template to assemble new strands
strand separation → strand elogation
chain elongation occurs 5’ → 3’
DNA vs RNA
DNA
double-stranded
contains deoxyribose
has adenine (A), thymine (T), guanine (G), and cytosine (C) as their nitrogenous bases
RNA
single-stranded
contains ribose
has adenine (A), uracil (U), guanine (G), and cytosine (C) as their nitrogenous bases
transcription & translation
transcription
takes place in nucleus
Helicase separates the 2 DNA strands
unwinds the double helix by breaking hydrogen bonds between complementary base pairs
only one of the 2 DNA strands will be used as a template to create the RNA
RNA polymerase binds to the template DNA at the promoter region and begins to synthesise RNA following the complementary bases
chain elongation occurs 5’ → 3’
translation
ribosome translates mRNA to amino acid
no direction in translation
read 5’ → 3’
DNA is read in sets of 3 nucleotides for each amino acid
a codon is a set of 3 ribonucleotides that code for an amino acid
there are 20 amino acids & 61 codons
therefore there are redundancies
a single amino acid may be coded for by more than one codon
the amino acid code must begin with the start codon
stop codons terminate the code
do not encode for amino acids
mutation
silent mutation
no change in amino acid sequence due to codon redundancies
protein synthesised is the same → same function
missense mutation
change in amino acid sequence
no change in protein length
protein folding might not fold properly → non-functional protein
nonsense mutation
change in protein length due to change in amino acid to nonsense codon
protein will not fold properly
protein will not work (non-functional protein)
frameshift mutation
nucleotide deleted / added to the sequence
causes the whole codon reading frame to move down or up causing all the amino acids to code wrongly
causes folding to be messed up
Cloning — Restriction, Digestion, Ligation
terminology
cloning —> making genetically identical copies of organisms, cells, or DNA
vector —> a piece of DNA that carries the gene of interest (goi) into the host cell
recombinant DNA —> a genetic segment from one organism is joined to a genetic segment from another to format hybrid molecule
competent cell —> bacteria that have undergone physical and/or chemical treatment so that they have enhanced ability to take up foreign DNA
steps in cloning
get the goi
purify DNA
usage of phenols and proteinases to remove protein
stick into vector
usage of a cloning vector (e.g. plasmids)
a piece of DNA that the goi will be pasted onto
carries the goi into the host cell
can self-replicate
ideal cloning vector
can accommodate goi
has a multiple cloning site (mcs)
easy cloning of goi
multiple re recognition sites
gives flexibility to the type of re that can be used to cut the goi
can self-replicate
has origin of replication (ori)
allows for self-replication
has antibiotic resistance gene
easy identification of host cells with vector
DNA ligase joins the double-stranded DNA fragments together using phosphodiester bonds
put vector into host cell
cell lysis
physical method
mortar and pestle
sonication
french press (that coffee thingy)
chemical method
NaOH + SDS
alkaline hydrolysis
enzymes
restriction
cutting the goi out using restriction enzymes (re)
bind to DNA strands at a specific sequence & cleave both strands
some have sticky ends (opposite: blunt ends)
extra nucleotides just hanging at the end of the strand w/o its complementary base pair
used to form hydrogen bonds with complementary base pairs of another strand of DNA
re should cut as close to the goi without cutting into it
different re produce different end sequences on the DNA fragments
goi and plasmid must be cut with the same re
only using 1 re will cause self-ligation
plasmid / goi closes into itself
chemical preparation of competent cells
recombinant plasmids added to 50 mM solution of $CaCl_2$
to remove negative charges of DNA
actual movement of DNA into competent cells is stimulated by heat shock (42ºC for 1 minute)
is super duper inefficient (1/1000 bacteria actually pick up the plasmid)
usage of antibiotic resistance property of plasmid to separate the transformed cells from the untransformed cells
using agar with antibiotic to weed out the untransformed cells
usage of insertional inactivation to pick out the cells with goi
example of LacZ gene
when undisrupted, LacZ gene produces β-galactosidase, which catalyses hydrolysis of X-gal to form a blue compound
when disrupted, LacZ gene cannot produce β-galactosidase, which will not catalyse hydrolysis of X-gal, therefore no blue compound is formed, causing the colony to appear white
Agarose Gel Electrophoresis
materials required
electrophoresis chamber & power supply
gel casting tray & comb
agarose gel
electrophoresis buffer (e.g. tris-borate-EDTA)
loading dye
DNA staining agent (e.g. SYBR green)
UV transilluminator
agarose
gelatinous substance
derived from polysaccharide that accumulates in the cell walls of red algae
melts at 85ºC and solidifies at 35ºC
agarose polymer in solidified gel forms a porous network
process of making the gel
molten gel loaded into the gel casting tray to set
comb put in to create wells for sample
solidified gel is submerged in electrophoresis buffer
electrophoresis buffer is an ionic solution with buffering capacity
used in gel runs to allow for current flow
loading dye is added to the sample
contains glycerol
allows the sample to sink into the well
must add DNA ladder to read the results
electro in electrophoresis
electrophoresis chamber is connected to the power pack
electric current causes DNA to move through gel
DNA carries a net negative charge
DNA flows towards the positive end of the electrode
shorter DNA move through the gel matrix faster
smaller fragments pass through pores more easily
staining the DNA
SYBR green used because non-toxic
ethidium bromide is better but its a carcinogen
binds to DNA
excited by 488 nm light, emits green fluorescence
analysis of electrophoresis
look at the number of bands
compare the bands to the DNA ladder
draw standard graph of the DNA ladder
distance travelled by log(kbp)
use SLAP-T to draw your perfect graph 😍
Spectrophotometry of DNA
principle
every substance has a maximum absorbance to a certain wavelength of light
absorbed light is converted into energy
unabsorbed light is reflected away from the surface
a spectrophotometer measures the amount of light that a sample absorbs
passes a beam of light through a sample
measures the intensity of the light reaching the detector on the other end
higher absorbance → higher optical density → more concentrated
blanking the spectro
there's a bunch of background readings
use the buffer to blank (not water unless water is your buffer)
DNA & RNA absorb 260 nm light
DNA —> 50 μg/ml of DNA gives OD reading of 1
conc. of undiluted DNA sample (μg/ml) = OD reading 50 dilution factor
RNA —> 40 μg/ml of RNA gives OD reading of 1
conc. of undiluted RNA sample (μg/ml) = OD reading 40 dilution factor
using 280 nm spectro for check for contamination
OD260/OD280 shld be between 1.8 and 2
<1.8 → protein contamination
2.0 → RNA contamination
Protein & Protein Quantification
proteins are made of amino acids
differ in terms of charges and hydrophobicity
depends on the side-chains
a polypeptide folds into a 3D functional protein
if a protein is denatured it doesn't work
change in shape of a protein (usually linearisation of protein)
can be caused by
salt concentration
pH
temperature
detergent
disulfide bridges, hydrogen bonds, ionic bonds, van der waals attraction, hydrophobic exclusion hold the 3D structure together
different proteins have different amino acid sequences
protein quantification
bradford assay
usage of coomassie blue dye
usually caps out at 465 nm
with protein caps out at 595 nm
need to make a protein standard
SLAP-T to the rescue 😍
fast, inexpensive and sensitive
protein-dependant (arginine)
incompatible with many detergents
coomassie blue dye stains quartz
best to use glass or plastic cuvettes
spectro at 280 nm
amino acids with aromatic rings absorb a bunch of light at 280 nm
concentration (mg/ml) = 1.55*A280
fast and convenient
no additional reagents required
no protein standard has to be prepped
high bias to tryptophan
can cause unreliability
only can use quartz cuvette
SDS-PAGE
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
separates proteins of different molecular sizes for analysis
similar concept to agarose gel but for proteins
unit for proteins is daltons (Da) [1 Dalton —> mass of 1 proton]
Materials
acrylamide & bis-acrylamide
acrylamide auto-polymerises into long straight chains of polymers
bis-acrylamide crosslinks the acrylamide polymers into a network
sodium dodecyl sulfate (SDS)
detergent to denature proteins to its primary structure
proteins are also boiled in the loading dye to denature them
loading dye contains glycerol to help sink the proteins into the wells
coats the amino acid so the protein has a net negative charge
TEMED & ammonium persulfate (APS)
APS provides free radicals to initiate polymerisation
TEMED is a catalyst to speed up the gel polymerisation reaction
gel casting tray & comb
electrophoresis chamber
proteins move from the cathode to the anode
smaller proteins pass through the pores of the acrylamide gel faster
coomassie blue
analysis similar to that of agarose gel (basically do the same thing)
SLAP-T once again !!
Size Exclusion Chromatography
column consists of microscopic beads
longer proteins pass through the column faster
shorter proteins get stuck in the beads and take longer to pass through the column
column must be washed thoroughly before use
after putting in the sample, wait for all the sample to be in the column before adding extra column buffer
count count count !!
each tube should have 5 drops (up till the last tube)
spectro at 280 nm
draw a bar graph of spectro to tube number (not histogram)
Polymerase Chain Reaction (PCR)
used to amplify DNA
materials
goi
primers (forward and reverse primers)
taq polymerase
each of the dNTP
thermocycler
steps
94ºC for 5 min
breaking hydrogen bonds between the 2 strands of DNA
32 cycles of
94ºC for 30 secs
denaturation
breaking hydrogen bonds between the 2 strands of DNA
68ºC for 30 secs
annealing
allow binding of forward and reverse primers
72ºC for 30 secs
elongation
allow synthesis of complementary strand