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What are the base pairs?
purine and pyrimidine
how many base pairs are there in E. coli?
4.6 million
how many base pairs are there in the human genome?
3 billion
Can RNA be complex?
Yes
the study of a gene often involves
Cloning it from its source using recombinant DNA technology to place it into a plasmid
The purification of genomic DNA can be used as a
Starting point for gene cloning
______ and ______________ allows researchers to go directly after the gene of interest from its source
PCR and the sequencing of the human genome
Most methods involve the purification of
Plasmid DNA and miniprep
DNA can be purified using
Organic solvents
DNA is stable at
Room temperature
Many protocols use proteinase K which
Degrades most proteins and is active in SDS, EDTA, urea, and a wide range of pHs
What are other reagents?
NaCl
Ethanol
Phenol
Chloroform
Centrifugation
What are all the DNA purification techniques?
Crude lysine
Anion Exchange
Cesium Chloride Gradient
Organic solvents
Alkaline Lysis
Silica-based methods
Crude lysine define
Heat cells, add proteinase K (not very clean)
Anion Exchange define
Backbone binds to + charged beads, alcohol precipitation
Cesium Chloride gradient define
High purity, but time-consuming
Organic solvents is also known as
Phenol-Chloroform Extraction
phenol-chloroform extraction define
phenol (aqueous, contains DNA and RNA), chloroform, isoamyl alcohol
Tedious, contains residual organic sand hazardous waste, may inhibit downstream exoperiments like PCR
Alkaline Lysis define
SDS: breaks up phospholipid belayer
NaOH: dissolves structural proteins
Agitation, precipitation, centrifugation
Silica-based methods
Selective absorption of DNA in the presence of chaotropic salts
Which two DNA purification techniques do we usually use?
Alkaline Lysis and Silica-based Salts
What does Alkaline Lysis involve?
Taking the bacteria and growing it overnight
1. resuspension
2. Lysis
3. Neutralization
4. Clearing of Lysates
At the end of Alkaline Lysis there is...
Silica-based column
In Phenol-Chloroform Expression adding PCIA
Separates out DNA
In Phenol-Chloroform Expression you can
Change the phase depending on pH
Cloned genes are usually stored in
Plasmids
Miniprep make how many micrograms of DNA?
10-30 micrograms of DNA
Miniprep produces
A decent amount of DNA in a short time
To get a pure plasmid you need to....
Take a colony and grow it overnight in a 2ml sugar. Spin it down and resuspend the pellet and add many buffers and a supernatant. The we wash the matrix and elute the DNA
How can you measure DNA quantification?
Spectrophotometer or Nanodrop
A260 of 1.0 =
50 ug/ml dsDNA
A260/A280 > 1.5 =
Pure
A230=
Organic contaminants
RNA is more susceptible to
Degradation than DNA
DNases require
Metal ions for activity and can be inactivate with EDTA, but RNases do not use metal ions
RNase is a
Single-strand specific endoribonuclease
RNase A is found in
Cells and tissues in high amounts
Also hands on to fight pathogens and in miniprep reagents
Resistance to metal agents
Can survive prolonged boiling or autoclaving
RNase activity is extremely difficult to
Inactivate
You also have to do what to work with RNase
Wear gloves
Bake glassware at 280 overnight
Add inhibitors or denaturing agents, such as b-ME, diethyl pyrocarbinate (DEPC), guanidium isothiocyanate, or recombinant ribonuclease inhibitor
mRNA may make up only
1-5% of total RNA
PolyA tail of mRNA binds to
Oligo-dT beads or columns
Example of an mRNA with a poly A Tail
mRNA - AUGUCUGAAAAA
Most kits are based off of
Chomczynski and Sacchi RNA Isolation
Chomczynski and Sacchi RNA Isolation
Uses TRIzol with contains phenol, chloroform, and guanidium isothiocyanate
DNA is in the....
RNA is in the....
proteins are at....
Organic phase
aqueous phase
Interphase
What is the pH levels in Chomczynski and Sacchi RNA Isolation
pH = 4.8 in organic phase, 7.5 for DNA purifications
what happens when rRNA and tRNA flow through the total RNA into the gel?
They form thick lines
What happens when mRNA flows through the PolyA RNA into the gel?
Creates a smear
What is needed to purify RNA?
Oligo magnetic beads
What binds to the Oligo magnetic beads to wash away the impurities?
Biotin
In RNA quantification
A260 of 1.0 =
A260:A280 > _____=pure
40 ug/ml
1.8
RNA can be quantified through other techniques...
Gel Electrophoresis
Northern blotting
RT-qPCR
What is the main way to quantify RNA?
RT-qPCR
Gel electrophoresis is frequently used to
Monitor orogress of many methods involving DNA
At alkaline
DNA, RNA, are negatively charged and migrate in an electric field
DNA has a built in
Charge to mass ratio
For small DNA fragments (<200 by) what can be used?
Polyacrylamide gel electrophoresis (PAGE)
For larger DNA molecules (>200 by) what can be used?
Agarose gel electrophoresis
What is agarose?
A polysaccharide from red seaweed
What does agarose do?
Readily polymerizes into a thick, porous gel
How much agarose is there typically?
1% (1 gram per 100 ml buffer)
The buffer is either ____ which is faster or _____ which offers better resolution
TAE, TBE
Agarose is added to TAE/TBE and ______________ ________ is added to solution
Ethidium bromide
The gel is poured into a coating frame and a ____________ is formed
Comb
Agarose is much thicker than
Acrylamide
DNA is loaded __________ to the current
Perpendicular
Is a stacking gel needed?
No
What does a tracking dye do?
Ensure they don't overlap with bands
DNAs isolectric point is ________, DNA has a high ______________, and migrates ____________ in the gel
5, charge-to-mass ratio, logarithmically
Gel contains ___________ of known molecular mass for comparison
Markers
The migration distance will be inversely proportional to
The log of the number of base pairs
Intensities can be compared to
Estimate a band's concentration (quanitification)
T/F: DNA can be linear, supercooled, or circular
True
T/F: It is easier to digest DNA ensure it is all linear for comparisons
True
What DNA conformation is the slowest and which is the fastest?
Nicked and circular are the slowest and supercool inning is the fastest
In pulsed-field electrophoresis standard electrophoresis is limited to
~50 kB
Large DNA fragments can be separated in an ____________ gel by using multiple electrodes
Agarose
RNA can be run on an agarose gel to check....
Sample's purity
Analyze ribosomal components
Verify RT-PCR products, or as a first step to or there blotting
Typically a _____________ used when RNA gel electrophoresis is used
RNA formaldehyde gel
There are several techniques available to visualize DNA and RNA, the simplest being
UV shadowing
Because Euclid acids absorb UV light (at 260 nm), passing light through the gel will result in
shadows
In UV shadowing there must contain a considerable amount of
DNA and RNA
What is high,y fluorescent when exposed to UV light?
Ethidium bromide
Facts about Ethidium Bromide
Can be added to the gel or soaked in afterwards
Nanogram detection
Must be discarded as hazardous waste
Methylene Blue and Crystal Violet allow for
Real-time visualization of DNA in gel (we can see results while stain is running)
Methylene Blue and Crystal Violet though is not
Very sensitive
Autoradiography can
Detect by exposing to X-Ray film, can expose gels for days, and provide to most sensitive detection
The genetic information of all living organisms is contained within the sequence of
nucleic acids
for most organisms, the genetic information is contained in
DNA, or RNA in some viruses
T/F: Proteins are unqie and high variable
True
What is now one of the easiest of the cellular molecules to analyze?
DNA Analysis
What new fields are used to study the human genome?
Genomics and Bioinformatics
In 1970 what did researchers invent?
DNA sequencing, Southern blotting, cloning, restriction enzymes, and many other essential tools still used today
Recombinant define
combining sequences that aren't normally found together and is the primary tool for genetic engineering of organisms
Example of recombinant
human + bacterial engineering
What does rDNA technoloy involve?
the biochemical manipulation of genes using enzymes that interact with DNA
What does the replication, transcription/expression, storage, maintanance, and repair of genome require?
many different enzymes that are useful in labratory
In the early days (1970s) DNA could be
melted and annealed, digested and ligated, and run on a gel
Researchers discovered valuable tools such as
vectors, restriction enzymes, DNA polymerase, DNA ligase, reverse transcriptase, ribonuclease, deoxyribonuclease, primase, topoisomerase, gel electrophoresis, clones, and more
DNA sequences from two different sources are spliced together to form a
replicon containing a gene of interest
