Polymerase Chain Reactions - Harrison High School

Gene Cloning

Definition


To clone a gene means to cut it out of its
original place in the host DNA and ligate it
into a suitable "vector" where multiple
copies of it can be quickly
reproduced.

How it works


1. Must attain a sufficient amount of the
DNA to be cloned.




2. Then attain a vector.


A vector is a piece of DNA that replicates in
bacterium.




How it works


3. Use of application restriction to cut the
DNA.


Next we use restriction enzymes to cut out the
gene we want from the amplified product. We
use the same enzymes to cut the vector so that
the ends will be compatible. This means that if
we use a "sticky ends" restriction enzyme, then
the DNA sequences of the trailing bits will be
compatible and will want to stick to each other.
This makes it much easier to do. If we can't find
a suitable site for a sticky end restriction enzyme
then we can use a "blunt
-
ended" one, which is
a bit harder to make work.


How it works


4. Ligation


Once both the vector and the target DNA
have been cut we mix them together and
add the ligase enzyme. This enzyme ligates
(connects) the
phosphodiester

backbone
acting as a glue to stick the ends together.

How it works


5. Transform bacteria


This just means add the DNA back into a
bacteria. There are a number of different
ways of doing this, but most of them involve
making the bacterial cell membrane
temporarily porous so that it can take up
the liquid containing the DNA mix. Then you
let the cells recover and grow them up as
usual.

How it works


6. Screening for 'positives'; Those bacteria
that have the cloned DNA in them.


Sometimes you don't get what you want!
For instance, a sometimes time the vector
will just stick back to itself, or stick to another
vector without having any target DNA in it.
So you have to have a mechanism for
picking out the ones where the target DNA
has gone into the vector.

Benefits


Combat diseases


Creates organs for transplants


Improve taste/quality of food


Repopulate endangered animals

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%20Cloning%20a%20Gene


Polymerase
Chain
Reactions

Sarah Schwartz, Haley
Kaufman,
Audine

Crosse

Definition/Overview

Polymerase Chain Reactions
:
A
mplifies a
single piece of DNA across several orders of
magnitude. The end result is the creation of
thousands to millions of copies of a
particular DNA sequence.

So how many steps are there?


It is a 3
-
step process (cycle) that is
repeated a specified number of times.
One PCR cycle consists of the following
steps:

1.
Denaturation

2.
Annealing

3.
Extension


The Steps


Denaturation
: Heat
separates double
-
stranded DNA into two
single strands.


Annealing
: Cool to
allow primers to form
hydrogen bonds with
ends of target
sequence.


Extension
: DNA
polymerase adds
nucleotides to the 3’
end of each primer.



So what happens next?


The process continuously repeats, forming
new molecules. For example, in cycle 1, 2
molecules are yielded. In cycle 2, 4
molecules are yielded. In cycle 3, 8
molecules are yielded.

Gel Electrophoresis


Used to
separate a mixed
population of DNA and RNA
fragments by
length, and to
separate proteins by
charge


Nucleic acid molecules are
separated by applying an electric
field to move the negatively
charged molecules through an
agarose matrix.


Sieving
–

Shorter
molecules move faster
and migrate farther than longer ones
because shorter molecules migrate
more easily through the pores of the
gel
.


Proteins are separated by charge in
agarose

Reading Gel Electrophoresis
Results


1
.) By looking at the migration of the
DNA molecular weight standards, you
can tell that the migration of DNA
through an
agarose

gel is not linear
with respect to
size.


If you graphed the distance
traveled vs. the molecular
weight of the fragment, you
would see that there is a
logarithmic relationship


Typically the samples are ran against a DNA ladder
which has DNA fragments of other known DNA sizes
so you can tell the size difference of the piece of
DNA in your sample.


The bands at the bottom are pieces of shorter DNA
and the faster they move through the gel. Towards
the top is the larger pieces of DNA and the slower
they move through the gel.

Gel
Electrophresis


Types of Gel


Agarose
-

Used to separate DNA fragments ranging from
50 base pair to several megabases. The distance between
DNA bands of a given length is determined by the percent
agarose in the gel. Gel setting is a physical, rather than
chemical change. Agarose gels do not have a uniform
pore size. Samples are also easily recovered.


Polyacrylamide
-

used for separating proteins ranging in size
from 5 to 2,000
kDa

due to the uniform pore size. Pore size is
controlled by controlling the concentrations of acrylamide
and
bis
-
acrylamide powder used in creating a gel.


Starch
-

Partially
hydrolysed

potato starch which is non
-
toxic. The gels are slightly more opaque. Non
-
denatured
proteins can be separated according to charge and size.
They are
visualised

using
Napthal

Black or
Amido

Black
staining. Typical starch gel concentrations are 5% to 10
%

Gel Electrophoresis


Gel Conditions


Denaturing
-

A denaturing gel is a type of
electrophoresis in which the native structure of
macromolecules that are run within the gel is not
maintained. In contrast to native, quaternary
structure cannot be investigated using this method.


Native
-

Native gel electrophoresis is an
electrophoretic separation method typically used in
proteomics and
metallomics
. Unlike denaturing gel,
native gel electrophoresis does not use a charged
denaturing agent
.

DNA Sequencing

By: Catherine Scott

Elizabeth
Dreisbach

Fallon
Prigmore

What is it?


Any process used to map out the sequence of the
nucleotides that comprise a strand of DNA


Any technology that is used to determine the order of
the four bases in a sequence of DNA: adenine, guanine,
cytosine, and thymine.


Adenine is paired with Thymine


Guanine is paired with Cytosine


DNA sequencing is a newer technology; It has been
known since the invention of the microscope that some
central part of the human cell has as its core some small
piece of information holding matter that probably
contains the blueprint of how each cell in your body is
form.

DNA Sequencing

Two common methods available are:


Maxam
-
Gilbert technique
which uses
chemicals to cleave DNA into fragments
at specific bases


The
Sanger Technique
uses DNA
polymerase to make DNA chains in the
presence of di
-
deoxynucleotides

to stop
the chain randomly as it grows.


This technique is more commonly used.


In both cases the DNA fragments are
separated according to length.

The Human Genome Project


The advent of DNA sequencing has
significantly accelerated biological
research and discovery.


It was devoted to developing and
bettering tools to make gene hunts faster,
cheaper, and more practical.


Produced the first complete sequences of
individual genome sequences.


As of 2012, thousands of human genomes
have been completely sequenced, and
many more have been mapped at lower
levels of resolution.

DNA structure and
sequencing

Animal
Cloning

Fun Facts


On July 5, 1996 Dolly the
sheep as the first successful
mammal cloned


The clones are only
genetically identical, they
can have different
personalities and physical
characteristics and
contains different
mitochondrial DNA
material


High failure rate


The animal cloning process is
called 'somatic cell nuclear
transfer'

Process of Reproductive
Cloning


Transfer of already made DNA into an
egg to make a genetically identical
organism


Steps


Nuclear Transfer


Stimulate Cell Division


Embryo Transplant in Host


Nucleus Transfer: transfers genetic
information from the animal cell to the egg
which has been stripped of its nucleus


Stimulated Cell Division: then the egg is
stimulated to divide by treatment with
chemicals or electric current


Totipotent: ability to create an entire
organism through cell division


Embryo Transplant in Host:


the cell begins dividing into a multi
-
cellular
embryo


It is planted into the uterus of a female host


The rest of the development occurs just like a
normal organism

Transformation


Maddie
, Meaghan, Al and
Gabriella

Definition


The insertion of a foreign gene into an
organism in order to change that
organism's expressed trait.




Natural Competence


The ability for cells to take up extracellular
genetic information


This state can occur as a result of
starvation or cell density

Plasmids

Circular molecules of genetic information
which replicate independently of bacterial
chromosomal DNA



Restriction Enzymes


Enzymes that protect the bacterial cell by
cutting up the foreign DNA from other
organisms or phages.

Glowing Tobacco


Bioluminescent gene from fireflies
uptaken

by tobacco






stem cells

Biotech
Applications

Addie, Kendall, Julia, Tai

Gene Cloning


Can be used to manipulate and analyze
DNA


In genetic engineering, bacterial
restriction enzyme are used to cut
molecules within short nucleotide
sequences, yielding a set of DNA
fragments


Cloning
vecotrs

include plasmids and bacterial artificial chromosomes.

Recombinant
plasminds

are returned to host cells, each of which divides

To form a clone of cells. Collections of clones are stored as genomic or

Complementary DNA libraries. Libraries can be screened for a gene of interest.

Using nucleic acid hybridization.

DNA Technology


DNA fragments separated by gel
electrophoresis


Short DNA fragments sequenced by
dideoxy

chain termination method


In humans, genome
-
wide association
studies use single nucleotide
polymorphisms as genetic markers for
alleles associated with particular
conditions

Stem Cell Research


Certain
embyronic

stem or adult stem cells
from animal embryos or adult tissues can
reproduce and differentiate in vitro as well as
in vivo, offering potential for medical use.


ES cells are pluripotent but difficult to acquire


Induced pluripotent cells resemble ES cells
and can be generated by reprogramming
differentiated cells. They hold promise for
medical research and
regenerative
medicine.