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Ch. 19
Part 2
Gene Technology
Vectors
• Using RE you have cut (or maybe
made) your desire gene
• How do you get it into organism?
• Must use a VECTOR (a go-between)
• Viruses
• bacteriophages
• Liposomes
• Tiny spheres of lipids containing DNA
• MOST Common vector is a PLASMID
• Small, circular piece of double-stranded
DNA
• Easy incorporation from prokaryotes
to eukaryotes
• Occur naturally in bacteria
• Replicated independently in bacteria
• Contain genes for antibiotic resistance
• Can be exchanged between bacteria
(during conjugation, even with different
species)
• If your gene is inserted in bacteria, you can
use bacteria to make your desired protein
from that gene (use the bacterial cell for
your benefit)
Plasmid Extraction and Gene Insertion
• Treat bacteria with your desired plasmid with
enzymes to break down cell wall
• Naked bacteria centrifuged
• Large bacteria chromosomes = heavy = sink to bottom
• Small chromosomes (plasmids) = lighter = remain on top
• Remove lighter (plasmid) DNA
• Cut circular DNA of plasmid using Restriction enzyme
• Must USE the same enzyme you used to cut out your
desired gene
• Creates the COMPLEMENTARY sticky ends (IMPORTANT in
integrating your desired DNA into plasma seamlessly)
•
If blunt ends are created, nucleotides with sticky ends must
be added to both gene AND plasmid in order to achieve
insertion
• Mix open plasmids and desired gene together
• Plasmid sticky ends pair up with sticky ends of desired
gene
• Add DNA ligase to the mixture
• Links together the sugar-phosphate backbone od desired
gene and plasmid
• Produces RECOMBINANT DNA = CLOSED circle of double
stranded DNA (plasmid with your desired gene inserted)
Why use plasmids?
• Plasmids can be modified to
produce excellent vectors
• Artificial plasmids can be created
• Example: pUC group of plasmids
• Low molecular mass
• Allows easy uptake by bacteria
• Origin of replication
• Easily copied
• Contain polylinker
• Several single target sites for different
restriction enzymes in one short
stretch of DNA
• short DNA sequence containing two
or more different sites for cleavage by
restriction enzymes
• Introduced into vectors to make
cloning easier
• Provide sites that allow cloning
DNA, cut with any of a number
of different restriction enzymes,
into a single plasmid
• One or more marker genes
• Allows for easy identification of cells
that have taken up plasmid
Getting Plasmids into Bacteria
• Treat bacteria with solution
containing HIGH concentration of
CALCIUM ions
• Cool solution
• Heat shock solution
• Increases chances of plasmids
passing through the surface of he
cell membrane
• Small proportion of bacteria (1%)
TRANSFORMED
• They have taken up the plasmid
• Remaining bacteria cannot be
used because:
• They have taken up taken up closed
plasmids and have NOT
incorporated them into their DNA
• They have not taken up plasmids at
all
Identifying Bacteria with Recombinant DNA
• In order to successfully make desired
proteins (gene product), you must find
and identify the bacteria that have
successfully incorporated the plasmid
into their DNA
• Processes of Identification
• Use of antibiotic resistance
• Not favored
• Use of Gene markers
• Favored method
Identification with Antibiotic Resistance
• Desired gene inserted into bacterial at the point
of a gene for antibiotic resistance
• Bacteria will no longer be resistant to the antibiotic
• Spread the bacteria on agar plate containing
antibiotic
• Any bacteria that has taken up the plasma will
NOT be resistant (will not grow on the plate)
• Those are the bacteria with successful incorporation of
the plasmid
• Bacteria’s DNA polymerase copies plasmids
• Bacteria divide by binary fission
• Each daughter cell has several copies of plasmid
• Each bacteria transcribes the protein (gene product)
made by the gene
• Problems:
• Spread of antibiotic resistant genes to other bacteria
(leads to untreatable diseases)
Insulin Production
• Diabetes Mellitus
• Caused by inability of pancreas to
make insulin
• Treatment
• Prior to 1980’sInsulin extract from
pigs/cattle and injected into patients
• 1983 human insulin made used GM
bacteria
• Problems Encountered
• Locating and isolating gene that
produced insulin in human DNA
difficult
• Cutting out gene for insulin too
difficult
Production of Human Insulin
• mRNA for insulin in pancreatic beta cells extracted
• Only cells expressing insulin gene
• Contain large quantities of insulin mRNA
• mRNA incubated with reverse transcriptase enzyme to produce single
stranded DNA
• Single stranded DNA converted to double-stranded DNA molecules with DNA
polymerase
• DNA molecule for insulin now inserted into plasmids of E. coli bacteria
• Advantages:
• Reliable supply to meet increasing demand for insulin
• Not dependent on animals
• Nucleotide sequence can be modified to create more effective insulin with different
properties
• Act faster
• Act slower
Reverse transcriptase
• Enzyme extracted
from RETROVIRUSES
• Reverses
transcription
(mRNA single
stranded DNA)
DNA Polymerase
• Enzyme
• Assembles
nucleotides to make
complementary DNA
strand
Genetic Markers
• a gene or DNA sequence with a known location on a chromosome
that can be used to identify individuals or species
• Use to identify transformed bacteria
• Bacteria that contain the plasmid with the inserted gene (that contains the
instructions to make our desired protein)
• Common Genetic Markers:
• Antibiotic resistance genes
• Less popular
• Lead to antibiotic resistant bacteria (dangerous)
• Genes that code for unique enzymes
• More popular
Useful Enzymes as Genetic Markers
• Enzymes that make protein GFP (green
fluorescent protein)
•
•
•
•
•
Obtained from jellyfish DNA
GFP fluoresces bright green in UV light
Gene for GFP is inserted into plasmids
Use UV light on bacteria
Whichever bacteria fluoresce bright green are the
bacteria that contain the transformed plasmid (the
genetically modified DNA)
• Enzyme B-glucuronidase (GUS)
• Obtained from E. coli bacteria
• Cells with this enzyme need to be incubated with
specific, colorless/non-fluorescent substrates
• They will transform this substrate into color of
fluorescent products
• Color/fluorescence helps identify activity of inserted
genes
• Color change = genetically modified organism
Promoters in Bacteria
• Not all bacterial genes are switched on
• Environmental conditions determine the
genes that bacteria will express
• Recall E.coli making the enzyme Blactosidase in presence of lactose medium
and no glucose (lac operon)
• Promoters control expression of genes
• Promoter  region of DNA where RNA
polymerase binds to begin transcription of
DNA
• What does this mean for our inserted genes
in the bacteria?
• We have to insert a promoter as well (to
ensure our gene is turned on)
• Promoter does TWO things:
1. Allows RNA polymerase to bind to DNA
2. Ensures that RNA polymerase recognizes
which of the 2 DNA strands is the template
strand
Promoters in Bacteria
• Promoter does TWO things:
1. Allows RNA polymerase to bind to DNA
2. Ensures that RNA polymerase recognizes which of
the 2 DNA strands is the template strand
• Promoter region of DNA contains TRANSCRIPTION STRAT
POINT
• First nucleotide of gene to be transcribed
• Promoter:
• CONTROLS the expression of the gene
• ENSURES high level of gene expression
• Eukaryotic Cell Transcription
• Transcription factors required to bind to promoter
region or RNA polymerase for transcription to start
Gel Electrophoresis
• method for separation and analysis of
macromolecules (DNA, RNA and proteins) and
their fragments, based on their size and charge
• Used in analysis of DNA and protein
• Involves:
• Agarose gel with “wells” in it
• Molecules placed in wells
• Electrical field applied to gel to make molecules move
through gel
• Movement of molecules depends on factors:
• Net (overall) charge
• Size of molecules
• Composition of gel
Gel Electrophoresis Factors
• Net (overall) charge
• Gel in chamber attached to voltage differential
• Negatively charged molecules move towards
POSITIVE anode
• Positively charged particles move towards NEGATIVE
anode
• Highly charged molecules move faster than less
charged molecules
• Size of molecules
• Small molecules move faster than large molecules
• Composition of gel
• Gel contains “pores”  tiny openings in which
molecules move through
• Size of pores determine speed which molecules
move through the gel
• Gel for DNA electrophoresis  AGAROSE
• Gel for protein electrophoresis  POLYACRYLAMINE
Gel Electrophoresis of Proteins
• Used to separate the polypeptides
made by different alleles of many
genes
• One gene can have many versions or
“alleles” (sections of DNA that code
for a specific protein)
• Proteins usually have an overall
NEGATIVE charge
• Proteins need to go through
IONIZATION process
• R-groups of amino acids become
charged
• Procedure needs to be carried out at
a constant pH using a buffer solution
Example of Protein Gel Electrophoresis
• Hemoglobin
• Many variants
• Adult hemoglobin  4 polypeptide
chains
• 2 alpha chains/ alpha globins
• 2 beta chains/ beta globins
• Sickle cell anemia
• Point mutation has caused a change in
the beta globin chain
• normal glutamic acid (polar R-group)
• after mutation  valine (non-polar Rgroup)
• Difference in charges of each polypeptide
chain means:
• Variants of beta globin can be separated
with protein electrophoresis
•
•
Sickle cell anemia polypeptide chain 
less negative charge, protein molecules
move SLOWER through the gel = shorter
distance traveled
Normal polypeptide chain  greater
negative charge, protein molecules
move FASTER through gel = farther
distance across gel
• Test used to find out if some carries
sickle cell allele
Gel Electrophoresis of DNA
• DNA structure
• Negatively charged phosphate group = overall negative charge
• SIZE of DNA is important
• Small fragments of DNA move fast
• Large fragments of DNA move slower
• Genetic Profiling/DNA fingerprinting is the application of gel electrophoresis of DNA and
forensic science
Steps of Gel Electrophoresis
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Make and use a gel (agarose for DNA, polyacrylamide for protein)
Use a comb when making gel to create “wells”
Wells should be made at the negative cathode end of the gel
Place samples of DNA into wells, using micropipette, changing tip for each
different sample to avoid cross-contamination
• Micropipette prevents movement fi DNA sideways/ensure DNA samples stay in
their specific well
•
•
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•
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Add stain/dye to DNA in each well
Add glycerin to sink DNA into well
Add buffer solution to gel in chamber (has specific pH)
Apply potential difference/voltage difference by turning on power supply
Stain and observe DNA
• Use UV light & DNA staining
• DNA samples are radioactive at beginning
• Use autoradiograph or X-ray to visualize
• Hazards and safety precautions:
• Electrical
• Chemical Irritants
• UV light
DNA Stains:
• Methylene blue
• Crystal violet
• Sybr green
• Ethidium bromide
• Acridine orange
• fluorescien
The t-Test
• Used to assess whether or not the MEANS of two sets of data with roughly
normal distributions are significantly different from each other
• Use when you want to know if two sets of continuous data are significantly
different from one another
• Criteria for using test:
• You have 2 sets of continuous, quantitative data
• Quantitative  numerical data collected from experiment
• Continuous  each measurement, count, or reading can be any value between two extremes
• Not necessarily whole numbers
• Discrete  each measurement, count, or reading can only be one of a set number of discrete values
• MUST be whole numbers
•
•
•
•
You have more than 10 but less than 30 readings of each set of data
Both sets of data come from populations that have normal distribution
Standard deviation for two sets of data is very similar
How to analyze results:
• Get your t-value
• If it is GREATER than the t-value for a probability of 0.05  significant difference
Standard Deviation Formula
𝑆=
𝑥−𝑥
𝑛−1
2
Step 3: calculate difference
between observation and
mean
Step 1: List each
x
observation
• 𝑥 = the mean
• Ʃ = “sum”
• x = number of
individual values
in a set of data
• n = total number
of observations
(individual values,
readings, or
measurements) in
one set of data
Step 2:
Calculate
• s = standard
the mean
deviation
(sum of all x
• Create a table!!!
values
divided by
total
amount of x
values)
𝑥−𝑥
𝑥−𝑥
1.1
-0.2
0.04
1.2
-0.1
0.01
1.3
0.0
0.0
1.4
0.1
0.01
1.5
0.2
0.04
1.4
0.1
0.01
1.1
-0.2
0.04
𝑥 = 9.0
n=7
𝑥 = 1.3
2
Step 4: calculate the
square root of each
difference
Step 5: calculate the
𝑥−𝑥
2
= 0.15
n–1=6
𝑥−𝑥
𝑛−1
2
SUM of all the square
differences
Step 6: Divide the
= 0.025
s = 0.158
SUM of all the square
differences by (n-1)
Step 7: Find the square
root to get the standard
deviation
The t-Test
• Start with a null hypothesis
• There is no significant difference between two sample of data
• Must have two sets of data
• Calculate mean of EACH set of data
• Calculate difference from the mean of all observations in each set of
data (𝑥 − 𝑥)
• Calculate the squares of each of the above (𝑥 − 𝑥)2
• Calculate the sum of the squares (𝑥 − 𝑥)2
• Divide by n1 – 1 for the first set and n2 – 1 for the second set
• n = number of individual measurements of sample set
• Take the square root of the above answer (standard deviation for each
set)
• Square the standard deviation and divide by the number of
observations in that sample, for both samples
• Add values together and take square root of the total
• Divide the difference in the two sample means with the value from the
previous step
• This is your t-value
• Calculate degree of freedom for all data (v)
• v = (n1 - 1) + (n2 - 1)
• Refer to table of t-values for your specific degree of freedom
• Probabilities in t-test table are probabilities that the null hypothesis IS CORRECT
(there is no significant difference)
• Probability you find is the probability that any difference in data is due to chance
𝑡=
𝑥1 − 𝑥2
𝑠12
𝑠22
𝑛1 + 𝑛2
𝑥1 = mean of sample data set 1
𝑥2 = mean of sample data set 1
s1 = standard deviation of sample 1
s2 = standard deviation of sample 1
n1 = number of individual measurements of sample 1
n2 = number of individual measurements of sample 1
t-Test
• Probaility of 0.05 is the critical value
• 5% confidence level
• Differences in data are due to chance if:
• T-test value represents a probability 0.05 or MORE
• Differences in data are NOT significant
• Number of observations IMPORTANT
• Total # of observations (both samples added together) is LESS than 30  error due
to chance is significant
• Table of t makes adjustment to critical values to take this into account
• This is why value of degrees of freedom needs to be calculated
• Total # of observations (both samples added together) is MORE than 30  number
of observations make little or no difference to critical values of t