Download Biogenetic Engineering & Manipulating Genes

Biogenetic Engineering &
Manipulating Genes
Chapter 20
Genomics video: Annenberg series
http://www.learner.org/resources/series187.html
Intro. Q’s #5 for Chapter 20: Genetic Engineering
1)
What does the acronym PCR stand for and what does this process
do?
2) What does Gel electrophoresis allow us to do?
3) Give two applications of DNA profiling.
4) What are the advantages and disadvantages of genetic screening?
5) How is a cDNA library different from a genomic library? (p. 388390)
6) Name two “vectors” that can be used for gene transfer.
7) Give two examples of a genetically modified crop or animal.
8) Briefly explain the process of gene therapy and give an example
how it works.
9) Explain what a clone is and how it could be formed.
10) What are some of the ethical concerns about cloning? Give your
opinion if you think cloning is something we should be doing.
Genetic Engineering
• Chapter 20
• DNA Technology
& Genomics
Pgs. 384-396 & 402-408
O.J. Simpson capital murder case,1/95-9/95
•
•
Odds of blood in Ford Bronco not being R. Goldman’s:
• 6.5 billion to 1
• Odds of blood on socks in bedroom not being N. Brown-Simpson’s:
• 8.5 billion to 1
Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J.
Simpson:
• 21.5 billion to 1
• Number of people on planet earth:
• 6.1 billion
• Odds of being struck by lightning in the U.S.:
• 2.8 million to 1
• Odds of winning the Illinois Big Game lottery:
• 76 million to 1
• Odds of getting killed driving to the gas station to buy a lottery ticket
• 4.5 million to 1
• Odds of seeing 3 albino deer at the same time:
• 85 million to 1
• Odds of having quintuplets:
• 85 million to 1
• Odds of being struck by a meteorite:
• 10 trillion to 1
Introduction
Manipulating genes and sequencing DNA has become one of the
greatest achievements in the last 50 years
-First complete genome sequenced was a bacteria (1995)
-Human Genome project (1990-2003)
Recombinant DNA
• Def: DNA in which genes
from 2 different sources are
linked
• Genetic engineering: direct
manipulation of genes for
practical purposes
• Biotechnology:
manipulation of organisms
or their components to
perform practical tasks or
provide useful products
Tools for DNA Analysis & Genomics
• PCR (polymerase chain
reaction)
• Gel electrophoresis
• Restriction fragment analysis
(RFLPs)
• Southern blotting
• DNA sequencing
• Human genome project
Tools of Genetic Engineering
• Restriction enzymes (endonucleases)
-in nature, these enzymes protect bacteria from intruding
DNA; they cut up the DNA (restriction); very specific
• Restriction site:
-recognition sequence for a particular restriction enzyme
• Restriction fragments:
-segments of DNA cut by restriction enzymes in a
reproducable way
• Sticky end:
-short extensions of restriction fragments
• DNA ligase:
-enzyme that can join the sticky ends of DNA fragments
• Cloning vector:
-DNA molecule that can carry foreign DNA into a cell and
replicate there (usually bacterial plasmids)
Restriction Enzymes (endonucleases)
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Producing Restriction Fragments
• DNA ligase used to splice together cut plasmids
and chromosome fragments
Cloning
Steps for Eukaryotic Gene Cloning
(pg. 387)
• Isolation of cloning vector (bacterial plasmid) &
gene-source DNA (gene of interest)
• Insertion of gene-source DNA into the cloning
vector using the same restriction enzyme; bind the
fragmented DNA with DNA ligase
• Introduction of cloning vector into cells
(transformation by bacterial cells)
• Cloning of cells (and foreign genes)
• Identification of cell clones carrying the gene of
interest
•
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Steps for Eukaryotic gene cloning
DNA Libraries (pg. 388)
• Collection of DNA fragments that have been
incorporated into plasmids
Genomic Libraries (pg. 388)
• Stored amount of cloned genes within bacteria or
virues (bacteriophages)
• Contained as cloned plasmids within bacteria or as
fragments within Bacteriophages genome
• Phages can carry a larger DNA insert vs. bacteria
cDNA Library (Pg. 390)
• cDNA (complementary DNA)
• Start w/mRNA to make a ssDNA strand
• Use an enzyme “reverse transcriptase” which is
found in retroviruses such as HIV (see pg. 340341) to make a ssDNA strand.
• A complementary strand of DNA is made by DNA
polymerase to produce a dsDNA molecule (cDNA)
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Polymerase Chain Reaction (PCR)
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
• Amplification of any piece of DNA without cells
(in vitro)
• Produces many identical copies of a DNA
segment
• Materials: heat, DNA polymerase, nucleotides,
single-stranded DNA primers
• Applications: fossils, forensics, prenatal
diagnosis, etc.
Polymerase Chain Reaction
• PCR = common method of creating copies of
specific fragments of DNA
– PCR rapidly amplifies a single DNA molecule into
many billions of molecules.
• Small samples of
DNA can produce
sufficient copies to
carry out
forensic tests
Polymerase Chain reaction (PCR)
Inserting DNA into the nucleus of a cell
Gel Electrophoresis (Lab)
• DNA fragments placed into “wells” in gel agarose
• Electricity pulls on DNA fragments
• Fragments travel at
different rates based
on size and ability to
squeeze through
swiss-cheese-like
agarose
Restriction Fragment Analysis
• Restriction fragment length polymorphisms (RFLPs)
• Southern blotting: process that reveals sequences and the
RFLPs in a DNA sequence
• DNA Fingerprinting (DNA Profiling)
Gel Electrophoresis
• separates nucleic acids or proteins on the basis of size and electrical charge
creating DNA bands of the same length
• DNA has a net negative charge (use a positive charge in the gel)
Intro. Q’s #5 for Chapter 20: Genetic Engineering
1)
What does the acronym PCR stand for and what does this process
do?
2) What does Gel electrophoresis allow us to do?
3) Give two applications of DNA profiling.
4) What are the advantages and disadvantages of genetic screening?
5) How is a cDNA library different from a genomic library? (p. 388390)
6) Name two “vectors” that can be used for gene transfer.
7) Give two examples of a genetically modified crop or animal.
8) Briefly explain the process of gene therapy and give an example
how it works.
9) Explain what a clone is and how it could be formed.
10) What are some of the ethical concerns about cloning? Give your
opinion if you think cloning is something we should be doing.
Video #3: Using DNA evidence
to Solve Crimes
• Write 15 key statements from the video.
• Please number each statement.
• Indicate where the sources of DNA were
found for each case.
O.J. Simpson capital murder case,1/95-9/95
•
•
Odds of blood in Ford Bronco not being R. Goldman’s:
• 6.5 billion to 1
• Odds of blood on socks in bedroom not being N. Brown-Simpson’s:
• 8.5 billion to 1
Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J.
Simpson:
• 21.5 billion to 1
• Number of people on planet earth:
• 6.1 billion
• Odds of being struck by lightning in the U.S.:
• 2.8 million to 1
• Odds of winning the Illinois Big Game lottery:
• 76 million to 1
• Odds of getting killed driving to the gas station to buy a lottery ticket
• 4.5 million to 1
• Odds of seeing 3 albino deer at the same time:
• 85 million to 1
• Odds of having quintuplets:
• 85 million to 1
• Odds of being struck by a meteorite:
• 10 trillion to 1
DNA Profiling (DNA fingerprinting)
Applications:
-Used in criminal investigations
-Identify the remains of dead people
-Compare Evolutionary origins of organisms
-Look for genetic disorders (mutations)
-Sequence DNA samples
DNA Fingerprinting
Southern Blot of DNA Fragments
(pg. 395)
• Used to determine the presence or absence of a
particular nucleotide sequence in the DNA
different sources.
• Compare the restriction fragments produced
from the different samples.
• Radioactive probes are used and attach to the
DNA fragments by complementary base pairing.
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Practical DNA Technology Uses
• Diagnosis of disease
• Human gene therapy
• Pharmaceutical products
(vaccines)
• Forensics
• Animal husbandry
(transgenic organisms)
• Genetic engineering in plants
• Ethical concerns?
Genetic Screening
Def: Testing individuals in a population for the
presence or absence of a gene (allele)
Advantages:
-pre-natal diagnosis of genetic disorders
-Could help stop the spread of a disorder
-Can detect carriers of a potential disorder
Disadvantages:
-invasion of privacy
-Individuals can become stigmatized in the
community
-Discriminated against or feared
-Employment and medical insurance
DNA Sequencing
• Determination of
nucleotide sequences
(Sanger method,
sequencing machine)
• Genomics: the study of
genomes based on
DNA sequences
• Human Genome
Project
Bacterial plasmids in gene cloning
Plant Cloning
• Tissue Culture Propagation
– Bits of phloem can
be induced in the
lab to form clumps
of tissue that will
make roots & shoots
–
Orchid culture
Embryo Cloning
• Medical technique which produces identical twins
or triplets
– Duplicates nature
– One or more cells are removed from a fertilized
embryo, encouraged to develop into one identical twins
or triplets
– Done for many years on
animals
– Limited experimentation
on humans
Adult DNA Cloning
• Cell nuclear replacement
–
–
–
–
Produces a duplicate of an existing animal
DNA from an embryo is removed;
Replaced with DNA from an adult animal
Embryo is implanted in a womb and allowed to develop
into a new animal
• Untried on humans potential of producing
a twin of an
existing person
Therapeutic Cloning
• Stem cells removed from an embryo with intent of
producing tissue or a whole organ for transplant
back into the person who supplied the new DNA
– Embryo dies in the process
• Goal is to produce a healthy
copy of a sick person's
tissue or organ for
transplant
Application: Gene Therapy
Therapeutic Cloning
• Vastly superior to organ transplants
– Supply would be unlimited - no waiting lists
• Tissue or organ would have the sick person's
original DNA
– No immunosuppressant
drugs would need to
be taken
Adult DNA Cloning
Adult DNA Cloning
Stem
cell
cloning
Theraputic Stem Cell Cloning
• Used
Chromatin
• Def: complex of DNA and proteins
• DNA Packing
• Histone proteins
(+ charged amino acids w/ phosphates of DNA
that are - charged)
• Nucleosome
-”beads on a string”; basic unit of
DNA packing
• Heterochromatin
-highly condensed interphase DNA
(can not be transcribed)
• Euchromatin
-less compacted interphase DNA
(can be transcribed)