Biotechnology and Genomicsprofamann.com/Site/Announcements/Entries/2014/11/23... · Biotechnology...
Transcript of Biotechnology and Genomicsprofamann.com/Site/Announcements/Entries/2014/11/23... · Biotechnology...
Biotechnology and Genomics_________________ is the production of identical copies of DNA, cells or organisms
If you think about it, members of a bacterial colony in a sample are clones because they all came from division of the same cell
____________ twins are even clonesSingle embryo separate to become two.
DNA of ���chromosome
Cell containing gene ���of interest
Gene inserted into���plasmid
Plasmid put into ���bacterial cell
Recombinant���DNA (plasmid)
Recombinant���bacterium
Bacterial ���chromosome
Bacterium
Gene of���interest
Plasmid
1
2
_______ ______ is production of many identical copies of the same gene.
If the inserted gene is replicated and expressed, we can recover the _______________or ________________.
HowIt’sDone
• Cuts DNA at specific points.• Cleaves vector (plasmid) and foreign (human) DNA. • Cleaving DNA makes DNA fragments ending in short single-
stranded segments with “______________”• These allow for insertion of foreign DNA into vector DNA.
5
A
T
A
T
A
T A
T
A
T
C
G
C
CG
G
C
G
A
T
A
T
A
T A
T
A
T
C
GC
CG
G
C
G
Google: McGraw-Hill “Restriction Endonucleases”
Like…
DNA Ligase
• Job?
• DNA “_____________” has now occurred
• The cell membranes of bacteria can be made more permeable to take up plasmids at this point.– Bacteria and plasmids will
reproduce.– Many copies of the plasmid and
many copies of the foreign gene are now produced.
6
Remember its function in DNA replication?
Google: mcgraw-hill: “Steps in Cloning a gene”
Go to www.sumanasinc.comAnimations-molecular biology-plasmid cloning
7
PCR
8
PCR cycles
DNA copies
first 1
second 2
third 4
old
old
old strand
new
new
new strand
DNA double strand
fourth 8 fi?h 16 and so forth
A three-step cycle—______________, ________________, ____________ —brings about a chain reaction that produces an exponentially growing population of identical DNA molecules
Go to www.sumanasinc.comAnimations-molecular biology-PCR
5ʹ′
Genomic DNA
TECHNIQUE
Cycle 1 ���yields
2molecules
Cycle 2 ���yields
4molecules
Cycle 3 ���yields 8
molecules; ���2 molecules ���
(in white ���boxes) ���
match target ���sequence
Target ���sequence
3ʹ′
3ʹ′
3ʹ′
3ʹ′
5ʹ′
5ʹ′
5ʹ′1
2
3
Advertisement for thermocycler (machine used for PCR)
http://www.cnpg.com/video/flatfiles/539/
Applications of PCR: Analyzing DNA Segments
• ________ ________________________ is the technique of using DNA fragment lengths called RFLP’s to match/investigate DNA samples.
• HOW?– By treating DNA samples with restriction
enzymes, a unique collection of different length fragments is produced
– Electrophoresing these fragments separates the fragments according to their• __________________
– Usually used to measure number of repeats of short sequences
– Produces distinctive banding pattern10
GOOGLE: MCGRAW - HILL “RESTRICTION FRAGMENT LENGTH POLYMORPHISM”
Electrophoresis is a laboratory technique for separating mixtures of charged molecules.
What is Electrophoresis?
• __________________: a material composed of two or more elements or parts.
• ___________________: a molecule (such as a protein or DNA) that has too many or too few electrons.
Charged molecules are separated based on their electrical charge and size.
Mixture of Charged
Molecules
• Law Enforcement Agencies
• Hospitals and Research
• Paternity Testing
• Biologists helping animals:
Zoos can use this technique to reduce possibilities of inbreeding. Taxonomists and evolutionary biologists can learn more about evolutionary relationships, identify species and document differences between them. Animals behaviorists can use DNA to glean information about the kinship of animals and how it impacts interactions with other animals
WHO USES IT?
Scientists can use this procedure to examine biological evidence found at crime scenes
Identify damaged genesIdentify viruses and bacterial genomesIdentify genetic diseases such as sickle cell anemia, Huntington’s disease, Duchenne’s muscular dystrophy,
Gene&cists can determine paternity as well as establishing gene&c links between large groups of people
• ___________________________– the flow of electric charge
• Acts as a _____________, a device to filter small particles out of a mixture of larger particles.
Essentials parts of GEL ELECTROPHORESIS
How Separation Occurs
Many molecules (amino acids, peptides, proteins, DNA, and RNA) have naturally occurring negative and positive charges on them. The sum of these charges determines the overall charge.
When introduced to an electrical current, negatively charged molecules are attracted to the positive electrode and positively charged molecules are attracted to the negative electrode.
-‐ +
-‐ +
-‐ + -‐
+ -‐
+
-‐ +
-‐ -‐ + -‐ -‐
-‐ -‐
Nega&vely Charged Protein
-‐ +
-‐ + +
Posi&vely Charged Pep&de
+
N
N
O +
Posi&vely Charged Amino Acid
Molecule Size:The porous material is made of microscopic particles suspended in a gel. The microscopic particles attach to one another forming tunnels that act as a sieve to separate the molecules. Small molecules can move faster than large molecules.
Porous Material
How Separation Occurs
Fig. 20-‐9a
Mixture of DNA mol-‐ ecules of different sizes
Power source
Gel
Anode Cathode
TECHNIQUE
1
2
Power source
– +
+ –
Homework: Do a virtual DNA fingerprin&ng exercise GOOGLE: NOVA + CREATE A DNA FINGERPRINT
Normal���allele
Sickle-cell ���allele
Large ���fragment
Electrophoresis of restriction fragments��� from normal and sickle-cell alleles
201 bp ���175 bp
376 bp
DdeI restriction sites in normal and��� sickle-cell alleles of β-globin gene
Normal β-globin allele
Sickle-cell mutant β-globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp175 bp
DdeIDdeI
DdeI DdeI DdeI DdeI
Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene
DNA Fingerprinting & Paternity
20
Mother Child Male 1 Male 2
Base re
peat units
many
few
DNA Band paQerns
Increasing size
b. Automated DNA fingerprinTng
a.
Fluo
rescen
ce units
Who is the father? Male 1 or 2?
GOOGLE:MCGRAW-HILL “DNA fingerprinting”
Go to www.sumanasinc.comAnimations-biotechnology-paternity testing
STR’s: short tandem repeats
21
A method of profiling called STR analysis uses loci in DNA where the same sequences of bases may occur a number of times.
For example the sequence GACT may be repeated 5x in one individual and 20x in another individual. The number of repeats varies from person to person
This can help identify individuals from each other or biological evidence
This is the FBI’s 13 Loci standard for matching individuals to evidence. The possibility of two individual’s coding the same in STR’s for the 13 loci is greater than 1 in a billion
Biotechnology Products• Genetically engineered organisms can produce
biotechnology products. They are called “genetically modified organisms” or (_______’s)
• Organisms that have had a foreign gene inserted into them are ______________.
22
Transgenic Bacteria• Genes of interest are inserted
into bacteria.• Bacteria are grown in large vats
called ________________ and product is harvested.– Products on the market include
insulin, hepatitis B vaccine, t-PA, and human growth hormone.
• Some genetic modifications can promote the health of plants and other can degrade harmful substances:
23
Transgenic Plants• Agricultural Crops– Foreign genes now give cotton, corn, and potato strains the
ability to produce an insect toxin– Soybeans are now resistant to a common herbicide – Weed-killer resisting crops are now common (“Roundup”
ready)
24
Human Hormone Production:
Transgenic Animals
• Vortex Mixing:– Many types of animal eggs
have taken up the gene for bovine growth hormone (bGH) through a procedure that uses agitation, tiny needles and foreign DNA to insert a gene into the egg.
– The procedure has been used to produce larger livestock animals
25
26
microinjection of human gene
.
.
milk
Genes coding for therapeutic & diagnostic proteins are incorporated into an animal’s DNA-The proteins appear in the animal’s
Gene Pharming:-Use of transgenic farm animals to produce pharmaceuticals
Plans are to produce drugs to treat:
Treating Disorders
! __________ ______ involves procedures to give patients healthy genes to make up for a faulty gene.
! It also includes the use of genes to treat genetic disorders and various human illnesses.
! There are __________ (outside body) and __________ (inside body) methods of gene therapy.
27
◦ In Vivo◦ Ex Vivo" Children with Severe Combined Immunodeficiency (SCID)
Human Genome Project
29
_________________ is the study of genomes of humans and other organisms.
_____________- All the genetic information of an individual (or species)
Goals of Human Genome Project
Did it ever get done? How many base pairs?
• Humans have between ____________ genes (still being researched)– Most genes code for proteins– Much of the other DNA was formerly described as “junk”
• “Junk” did not specify the order of amino acids in a polypeptide
30
Recent observations suggest that between 74% and 93% of the genome is transcribed into RNA but not all translated into proteins
Therefore, the “junk” (nontranslated RNA) may code for regulatory gene expression necessary for proper cellular events.
Gene Structure ! Historically, genes were
defined as discrete units of heredity that corresponded to a locus on a chromosome.
! Prokaryotes typically possess a single circular chromosome without ____________ and have less DNA.
! Eukaryotic chromosomes with much more DNA are much more complex.
31
Eukaryotic Gene Structure• ___________________________ are DNA sequences
that occur between genes• __________________ DNA elements occur when the
same sequence of two or more nucleotides are repeated many times along the length of one or more chromosomes.
• __________________ are specific DNA sequences that have the remarkable ability to move within and between chromosomes. AKA: jumping genes!
32
Remember STR’s?... Can also be referred to as ______________
YouTube: Transposons: Shifting segments of the genome
• The study of the structure, function, and interaction of cellular proteins
• At least 20-25K of our genes are translated into proteins
• The sum total of these is called the human __________
• Understanding protein function is essential to the development of better drugs. How?
• Once the primary structure of these protein is known– It should be possible to predict their tertiary structure– Computer modeling of the tertiary of these proteins is an important part of
proteomics
33Worth a look -one last link for biotech products from BIO-RAD:http://bio-rad.cnpg.com/Video/flatFiles/799/
Current understanding of what a “Gene” is…
• A genomic sequence (either DNA or RNA) directly encoding functional products, either RNA or protein.
34