Genetic Technology - Teacher

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22 Οκτ 2013 (πριν από 3 χρόνια και 9 μήνες)

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Genetic Technology

Biotechnology


Traditional vs. Modern Biotechnology


History of Biotechnology


Ethical issues

Genetic Engineering


Involves cutting (or cleaving) DNA from
one organism into small fragments and
inserting the fragments into a host organism
of the same or a different species


AKA: Recombinant DNA technology


Recombinant DNA is made by connecting, or
recombining, fragments of DNA from different
sources

Transgenic Organisms


Contain recombinant DNA


Examples: Glowing tobacco (pg 349)





Bt corn





Golden rice


Steps involved in creating a
transgenic organism


Step 1: Isolate the DNA
fragment that will be inserted
(use a restriction enzyme)



Step 2: attach the DNA
fragment to the “vehicle”
(virus or bacteria DNA) by
“gene splicing”



Step 3: transfer the vehicle
into the host organism
(recombined DNA is
transferred to a bacterial cell)

Uses of Recombinant DNA


Industry


Medicine


Transgenic animals


Agriculture


Transgenic plants

A Revolution in Biological
Science


In mid
-
1970s, newly developed
recombinant DNA technology

provided, for the first
time, powerful techniques for studying and manipulating DNA


Recombinant DNA technology allows biologists to redirect the genetic activity of
organisms


Techniques and approaches include

1.
Splicing:

Specific genes can be added or removed by cutting and rearranging
DNA

2.
Genetic engineering:

Modification of the DNA of an organism to produce new
genes

3.
DNA cloning:

Large quantities of DNA can be obtained relatively easily by
cloning cells and amplifying specific DNA sequences, both
in situ

(inside) and
in vitro

(out of) the cell

4.
Biotechnology
, the overall corporate
-
driven use of genetic engineering, which
already has had great impact on our lives


History and Terminology


Biotechnology has its roots in
microbiology
: the study of micro
-

organisms (usually bacteria)


Bacteriophage

(viruses of bacteria) were first used to try to
understand how DNA worked (recall Hershey
-
Chase)


Scientists learned how to make bacteria
competent

for transformation
(recall Griffith) by modification of the ionic environment…


Made the cell wall more permeable


Allowed the cells to take up DNA


Genetic engineering not possible prior to discovery of
restriction
endonucleases

(
restriction enzymes
) by Ham Smith and Daniel
Nathans (Johns Hopkins


Nobel Prize winners


1978)


Specifically clip strands of DNA


Many different types






Restriction Enzymes 1


Restriction enzymes are natural, bacterial “molecular scissors”
normally used to destroy non
-
host (such as bacteriophage) DNA


Cut DNA at specific base pair sequences: many are
palindromic


A
linguistic palindrome

has the same informational sequence forward and
backward


MadamImadam is a linguistic palindrome


A
nucleic acid palindrome

has the same sequence on two antiparallel,
complementary, hydrogen
-
bonded strands


e.g. AACGTT will pair with TTGCAA; these are palindromes


Restriction enzymes cut at the ends of the palindromic sequences (red
in the example here)


They are cut in a staggered fashion:


Restriction Enzymes 2


Restriction enzymes snip the phosphodiester bond at a very VERY
specific location based on the sequence information


Staggered cuts on ends of palindromic regions leave strands with
complementary “sticky” ends when separated (usually

with heat)






Segments of DNA with sticky ends can hydrogen bond with
complementary sequences


The open spaces can be joined with purified naturally
-
occurring DNA
ligases ( )


process is called
splicing


L

L

New seq.

New seq.

(complementary)

L

Heat denaturation

Vector DNA


A
vector

is a genome that carries foreign DNA into a host
cell


Used to transform
competent

(can take up DNA) bacteria


Bacteriophage (bacterial viruses


recall Hershey
-
Chase)


Can carry DNA segments of up to 15kb


Engineered mammalian viruses used in mammalian cells


DNA incorporated into nuclear DNA of mammalian cell


Plasmids are small rings of double
-
stranded DNA that commonly
occur in bacteria


Can carry DNA segments < 10kb in size 1Kb=1000 bps


Often carry genes for resistance to antibiotics


Can be used to provide a selectable marker which allows only transformed
cells to live. Cells containing ampicillin resistance gene inserted by
transformation can be grown on ampicillin
-
rich media; nontransformed cells
die

Plasmids and Bacteria



Recombinant DNA Is Formed

by Splicing DNA From a Vector

Into Host DNA



PCR Is Used to Amplify DNA
in Vitro


The

Polymerase Chain Reaction (PCR)

allows amplification of a
small amount of targeted DNA in a short time. It is very simple but
very powerful.


PCR has three steps:

1.
Denaturation.

A buffered mixture of primers, nucleotides,
Taq

polymerase and DNA fragments is heated to dissociate ds DNA
into ssDNA

2.
Annealing of primers.

The solution is cooled and the primers
bind to complementary sequences of the DNA at the ends

3.
Primer Extension.

DNA polymerase then uses the nucleotides to
extend and make more copies of each strand


The process is repeated over and over to produce millions of copies of
the original DNA strand

PCR


PCR Characteristics


DNA
Taq

polymerase isolated from the thermophilic bacterium
Thermus aquaticus

is used as it is not damaged by the heat


After 20 cycles a single fragment produces more than one million
(2
20
) copies


30 cycles will produce a billion times the original amount (2
30
),
enough amplification to reveal the presence of a single copy of a
specific
target sequence


The use of PCR is virtually limitless


Criminal investigations (DNA fingerprints) from a speck of blood
or single hair


Detection of genetic defects in very early embryos by collecting a
few sloughed
-
off cells from the amniotic fluid (amniocentesis) and
amplifying the DNA


Used to examine historical figures and extinct species such as
mammoths and dodos


Very sensitive and samples easily contaminated

Gel Electrophoresis Is Widely Used
to Separate DNA and RNA


DNA and RNA are negatively charged, and move through a gel at
varying speeds due to different molecular lengths (sizes)


Restriction endonucleases can be used to clip the DNA


DNA fragments are loaded on a gel & an electric field is applied


Bigger DNA fragments migrate through the gel more slowly than
small fragments


Fragments can be stained and visualized migrating as bands under UV
light


DNA fragments can be transferred (‘blotted’) to a filter, denatured and
incubated with a radioactive or fluorescent probe which will hybridize
to the target sequence and be revealed by autoradiography or sensitive
color digital camera

Southern and Northern
Blotting


Blots for DNA are called Southern blots


Named after its inventor, E.M. Southern (1975)


1.
DNA is separated on a gel

2.
Gel is transferred onto nitrocellulose or a nylon membrane

3.
Membrane is incubated with radioactive ssDNA probe of the gene of
interest

4.
Probe hybridizes to the blot where there is a fragment with a
complementary sequence

5.
The radioactive bands on the blot identify fragments of interest

6.
If RNA blotted, called Northern blot

Western Blotting

1.
Proteins separated in a gel

2.
Proteins blotted onto a membrane

3.
Antibodies specific for a particular protein
are applied

4.
Antibodies stick to target proteins ONLY

5.
Revealed by additional antibodies attached
to enzymes that precipitate a colored
product

DNA Sequences Contain Much
Information


Can determine the actual protein encoding regions…
the ORFs


Regions containing transcriptional signals and RNA
processing can be recognized


Amino acid sequences of proteins can be inferred
from the base sequence


much faster and easier
than from the protein directly


Reveals structure of chromosomes, possibly helpful
in determining evolution, phylogenies, and fighting
disease

DNA Nucleotide Sequencing

1.
Radioactive DNA is replicated off the host template DNA

2.
Dideoxynucleotides (ddNTPs


lacking OH at 3’ and 2’)

are
incorporated in small quantities in the reaction mixture to label
sequences which contain those deoxybases (the ddNTPs jam DNA
polymerase)

3.
Reaction mixtures contain DNA polymerase, radioactive primers,
single
-
stranded DNA fragment, 4 deoxynucleotides. Four tubes are
prepared


each containing a different dideoxynucleotide (ddATP,
ddCTP, ddGTP or ddTTP)

4.
Fragments of varying length are formed in each mixture


the end
points occur at the 4 different ddNTP

5.
Fragments are separated based on length by electrophoresis

6.
Autoradiography reveals the presence of the radioactively
-
labeled
DNA fragments

7.
The DNA sequence is literally ‘read off’ of the gel, using the 4 lanes
derived from the 4 reaction tubes.



Restriction Fragment Length

Polymorphism Analysis


Each individual carries with it a record of the variation in
genetic organization from its previous generations. There
is a LOT of variation in individuals of a population


Restriction enzymes are used to cut DNA into fragments


The fragments are of different lengths in different
individuals since each host DNA is unique. When three
different individuals DNA are cut with a restriction
endonuclease, 3 different fragment sizes are likely to be
produced, unless they are identical triplets

An RFLP
Autoradiogram


A ‘DNA fingerprint’
produced by gel
electrophoresis reveals
different banding patterns


restriction fragment length
polymorphisms (RFLPs)


This technology is particularly
important in determination of
paternity and in forensics


Here, M = mother, F = father,
and C = children. Note that
children have all bands of M
and F lanes.