chapter 3 recombinant dna technology and genomics
TRANSCRIPT
Chapter 3Chapter 3
Recombinant DNA Technology and Genomics
Agarose Gel ElectrophoresisAgarose Gel Electrophoresis
Electrophoresis is a molecular technique that separates nucleic acids and proteins based on:
Size
and
+-+Charge+-+
DNA is a negatively charged molecule and therefore is attracted to positive charges.
Agarose Gel ElectrophoresisAgarose Gel Electrophoresis
Agarose provides a matrix through which DNA molecules migrate. Larger molecules move through the matrix
slower than small molecules The higher the concentration of agarose, the
better the separation of smaller molecules
Agarose Gel ElectrophoresisAgarose Gel Electrophoresis
How to make an agarose gel:1. Weigh out a specified amount of agarose
powder.2. Add the correct amount of buffer.3. Dissolve the agarose by boiling the solution.4. Pour the gel in a casting tray.
5. Wait for the gel to polymerize.
Agarose Gel ElectrophoresisAgarose Gel Electrophoresis
How to make an agarose gel:6. Place gel in chamber and cover with buffer7. Add loading dye to the sample
8. Load sample on to the gel.
Agarose Gel ElectrophoresisAgarose Gel Electrophoresis
How to make an agarose gel:9. Stain the gel10.Take a picture of the gel11.Analyze results
Agarose Gel ElectrophoresisAgarose Gel Electrophoresis
Agarose Gel ElectrophoresisAgarose Gel Electrophoresis
Electrophoresis Animation
Recombinant DNARecombinant DNA
Recombinant DNA technology Allows DNA to be combined from different sources Also called genetic engineering or transgenics
Vector – DNA source which can replicate and is used to carry foreign genes or DNA fragments.
Recombinant DNA – A vector that has taken up a foreign piece of DNA.
Recombinant DNARecombinant DNA
Restriction enzyme – an enzyme which binds to DNA at a specific base sequence and then cuts the DNA Restriction enzymes are named after the bacteria from which they were isolated.
• Bacteria use restriction enzymes to chop up foreign viral DNA
Restriction EnzymesRestriction Enzymes
Recognition site – specific base sequence on DNA where a restriction enzyme binds.• All recognition sites are palindromes, which means
they read the same way forward and backward.example: RACECAR or GAATTC CTTAAG
• Each restriction enzyme has its own unique recognition site.
Restriction EnzymesRestriction Enzymes
Restriction Enzymes
After cutting DNA with restriction enzymes, the fragments can be separated on an agarose gel.• The smaller fragments will migrate further than the
longer fragments in an electric field.• The bands are compared to standard DNA of known
sizes. This is often called a DNA marker, or a DNA ladder.
Restriction EnzymesRestriction Enzymes
Restriction Enzymes
Running a Restriction digest on an agarose gel
Restriction Enzymes
After analyzing your results, you draw a restriction map of the cut sites.• A restriction map is a diagram of DNA showing
the cut sites of a series of restriction enzymes.
Restriction Enzymes
Restriction Enzymes
Most restriction enzymes cut within the recognition site.
When restriction enzymes cut in a zig zag pattern, sticky ends are generated.
Restriction EnzymesRestriction Enzymes
Overhanging sticky ends will complementarily base pair, creating a recombinant DNA molecule.
DNA ligase will seal the nick in the phosphodiester backbone.
Restriction EnzymesRestriction Enzymes
Restriction EnzymesRestriction Enzymes
Restriction Enzyme Animation
TransformationTransformation
Transformation – the process by which organisms take up and express foreign DNA
Griffith’s experiment
Bacterial Transformation Bacteria, such as E.coli, can take up and
express foreign DNA, usually in the form of a plasmid.
TransformationTransformation
Gene cloning – using bacterial transformation to make lots of copies of a desired gene.
TransformationTransformation
Gene Cloning Animation
Transformation
Steps of Bacterial Transformation1. Choose a bacterial host
a. E. coli is a model organismi. Well studiedii. No nuclear membranesiii. Has enzymes necessary for replicationiv. Grows rapidly (20 min. generation time)v. Inexpensivevi. Normally not pathogenicvii. Easy to work with and transform
TransformationSteps of Bacterial Transformation
2. Choose a plasmid to transforma. Characteristics of a useful plasmid
i. Single recognition site• Plasmid only cuts in one place, so this ensures that the
plasmid is reformed in the correct order.
ii. Origin of replication• Allows plasmid to replicate and make copies for new cells.
iii. Marker genes• Identifies cells that have been transformed.
gene for antibiotic resistance – bacteria is plated on media with an antibiotic, and only bacteria that have taken up a plasmid will grow
gene that expresses color – bacteria that have taken up a recombinant plasmid are a different color than bacteria that have taken up a NONrecombinant vector.
Transformation
Steps of Bacterial Transformation3. Prepare bacterial cells for transformation
a. Treat with calcium chloride – softens the phospholipid bilayer of the cell membrane, which allows the plasmid to pass through
b. Electroporation – brief electric pulsec. Directly inject plasmid into bacterial host
Transformation
Steps of Bacterial Transformation4. Plate transformation on
appropriate mediaa. Contains nutrients for bacteria and
antibiotic to distinguish transformed bacteria from NONtransformed bacteria
5. Incubate plates overnighta. E.coli grows at body temp. (37 °C)
6. Analyze plates
Gene Cloning Animation
Gene Cloning
What makes a good vector?
Gene Cloning
What makes a good vector?
Gene Cloning
How do you identify and clone a gene of interest?• BUILD A LIBRARY!!
• DNA library – a collection of cloned DNA fragments from a particular organism
• Can be saved for a relatively long period of time and screened to pick out different genes of interest
• Two types of libraries1. Genomic library – contains DNA sequences from entire genome2. cDNA library – contains DNA copies of mRNA molecules expressed
Construction of a DNA library Animation
Gene Cloning
Gene Cloning
Steps to screen a library1. Plate cells and transfer to
nylon membrane2. Lyse bacterial cells3. Denature DNA4. Add radioactively labeled
probe that is complementary to gene of interest
Gene Cloning
Steps to screen a library5. Wash off non-specifically
bound probe6. Expose membrane to x-ray
film7. Align exposed film with
original plate8. Grow cells containing gene of
interest in culture.
Gene Cloning
Rarely is an entire gene cloned in one piece, even in a cDNA library, therefore must “walk” the chromosome until a start and stop codon are found.
SequencingSequencing
Sequencing – determining the order and arrangement of G’s, A’s, T’s and C’s in a segment of DNA.
Let’s review replication…..
SequencingSequencing
The Sanger sequencing method uses dideoxy-nucleotides to generate all possible fragments of the DNA molecule to be sequenced.
SequencingSequencing
deoxynucleotide dideoxynucleotide
Set up four different reactions:
SequencingSequencing
Load the four reactions in different wells of a polyacrylamide gel to separate the fragments
SequencingSequencing
SequencingSequencing
Sequencing Animation
Human Genome Project
Initiated in 1990 with plan to identify all human genes• Analyze genetic variation
among humans• Map and sequence genomes
of model organisms• Develop new lab technology• Disseminate genome
information• Consider ethical, legal, and
social issues that accompany genetic research
Human Genome Project
Francis Collins Craig Venter
Human Genome Project
Consider ethical, legal and social issues• Who owns your DNA?
Develop new lab technology• Automated Sequencing
Human Genome ProjectHuman Genome Project
Human Genome Project
Disseminate genome information• GenBank database
Human Genome Project
Analyze genetic variation among humans• The genome is approximately 99.9% identical
between individuals of all nationalities and backgrounds.
Human Genome Project
Map and sequence genomes of model organisms• E.coli
• Arabidopsis thaliana
• Saccharomyces cerevisiae
• Drosophila melangaster
• Caenorhabditis elegans
• mus musculus
PCR
Polymerase chain reaction (PCR) A lab technique used to amplify segments of DNA
"PCR has transformed molecular biology through vastly extending the capacity to identify, manipulate and reproduce DNA. It makes abundant what was once scarce -- the genetic material required for experimentations."
PCR
Reaction requirements Template DNA – total genomic
DNA isolated from an organism that contains a target region to be amplified
DNA primers - Short pieces of single stranded DNA that flank the target
Taq DNA polymerase - Attaches nucleotides on the growing strand of DNA
Nucleotides (GATC) – Polymerase adds complementary nucleotides to the template
PCR
Reactions are placed in a machine called a thermal cycler. The machine cycles through three temperatures.
PCR
1. Heat samples to 94°C for a minute or so to denature the double stranded template DNA.
PCR
2. Drop temperature to around 50 or 60°C to allow primers to anneal.
PCR
3. Maintain temperature at 72°C for a minute or two to allow the polymerase to elongate the new DNA strands.
PCR
The thermal cycler repeats the denaturing, annealing, and elongating temperatures approximately 30 times.
PCR Animation
PCR
PCR amplification is logarithmic, meaning the number of copies of the target is doubled every cycle. (2n)
PCR
PCR animation
PCR
Cloning by PCR• Design primer specific for gene of interest
(must know some of the sequence)• Can use a T-vector because Taq polymerase
adds an A to the 3’ end of sequence
Applications of Recombinant DNA Technology
Chromosomal Location and Gene Copy Number
Fluorescence in situ hybridization (FISH)
Southern Blot - molecular technique where DNA is transferred onto a membrane from an agarose gel and a probe is hybridized.
Chromosomal Location and Gene Copy Number
Southern Blot
The first step in preparing a Southern Blot is to cut genomic DNA and run on an agarose gel.
Southern Blot
The next step is to blot or transfer single stranded DNA fragments on to a nylon membrane.
The next step is to hybridize a radioactively labeled DNA probe to specific sequences on the membrane.
Southern Blot
Southern Blot
The last step is to expose the radioactively labeled membrane to a large sheet of film.
You will only visualize bands where the probe hybridized to the DNA..
Southern Blot Animation
Southern Blot
Studying Gene Expression Northern Blot
• Isolate RNA from tissue of interest• Separate on agarose gel• Blot onto nylon membrane• Hybridize probe specific for desired
transcript• Expose on film
Reverse Transcription PCR (RT-PCR)• Used if RNA produced is below
detection level for Northern blot• Isolate RNA from tissue of interest• Convert into double stranded cDNA• Amplify by PCR• Run on agarose gel
Studying Gene Expression
Real Time PCR (qPCR)• Eliminates the need
for running agarose gels
• Is quantitative
Studying Gene Expression
Gene microarray
Microarray animation