EXAM 3 UCF QBM Dr. Borgon

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