Title: Recombinant DNA Technology and Genomics
1Chapter 3
- Recombinant DNA Technology and Genomics
2Agarose Gel Electrophoresis
- Electrophoresis is a molecular technique that
separates nucleic acids and proteins based on - Size
- and
- -Charge-
Shape
3Agarose Gel Electrophoresis
- DNA is a negatively charged molecule and
therefore is attracted to positive charges.
4Agarose 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
5Agarose Gel Electrophoresis
- How to make an agarose gel
- Weigh out a specified amount of agarose powder.
- Add the correct amount of buffer.
- Dissolve the agarose by boiling the solution.
- Pour the gel in a casting tray.
- Wait for the gel to polymerize.
6Agarose Gel Electrophoresis
- How to make an agarose gel
- Place gel in chamber and cover with buffer
- Add loading dye to the sample
- Load sample on to the gel.
7Agarose Gel Electrophoresis
- How to make an agarose gel
- Stain the gel
- Take a picture of the gel
- Analyze results
8Agarose Gel Electrophoresis
Electrophoresis Animation
9Recombinant DNA
- Recombinant DNA technology
- Allows DNA to be combined from different sources
- Also called genetic engineering or transgenics
10Recombinant DNA
- 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.
11Restriction Enzymes
- 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
12Restriction 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.
13Restriction Enzymes
14Restriction 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.
15Restriction Enzymes
- Running a Restriction digest on an agarose gel
16Restriction 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.
17Restriction Enzymes
18Restriction Enzymes
19Restriction Enzymes
- Most restriction enzymes cut within the
recognition site. - When restriction enzymes cut in a zig zag
pattern, sticky ends are generated.
20Restriction Enzymes
- Overhanging sticky ends will complementarily base
pair, creating a recombinant DNA molecule. - DNA ligase will seal the nick in the
phosphodiester backbone.
21Restriction Enzymes
Restriction Enzyme Animation
22Transformation
- Transformation the process by which organisms
take up and express foreign DNA
Griffiths experiment
23Transformation
- Bacterial Transformation
- Bacteria, such as E.coli, can take up and express
foreign DNA, usually in the form of a plasmid.
24Transformation
- Gene cloning using bacterial transformation to
make lots of copies of a desired gene.
Gene Cloning Animation
25Transformation
- Steps of Bacterial Transformation
- Choose a bacterial host
- E. coli is a model organism
- Well studied
- No nuclear membranes
- Has enzymes necessary for replication
- Grows rapidly (20 min. generation time)
- Inexpensive
- Normally not pathogenic
- Easy to work with and transform
26Transformation
- Steps of Bacterial Transformation
- Choose a plasmid to transform
- Characteristics of a useful plasmid
- Single recognition site
- Plasmid only cuts in one place, so this ensures
that the plasmid is reformed in the correct
order. - Origin of replication
- Allows plasmid to replicate and make copies for
new cells. - 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.
27Transformation
- Steps of Bacterial Transformation
- Prepare bacterial cells for transformation
- Treat with calcium chloride softens the
phospholipid bilayer of the cell membrane, which
allows the plasmid to pass through - Electroporation brief electric pulse
- Directly inject plasmid into bacterial host
28Transformation
- Steps of Bacterial Transformation
- Plate transformation on appropriate media
- Contains nutrients for bacteria and antibiotic to
distinguish transformed bacteria from
NONtransformed bacteria - Incubate plates overnight
- E.coli grows at body temp. (37 C)
- Analyze plates
Gene Cloning Animation
29Gene Cloning
- What makes a good vector?
30Gene Cloning
- What makes a good vector?
31Gene 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 libraries
- Genomic library contains DNA sequences from
entire genome - cDNA library contains DNA copies of mRNA
molecules expressed
Construction of a DNA library Animation
32Gene Cloning
33Gene Cloning
- Steps to screen a library
- Plate cells and transfer to nylon membrane
- Lyse bacterial cells
- Denature DNA
- Add radioactively labeled probe that is
complementary to gene of interest
34Gene Cloning
- Steps to screen a library
- Wash off non-specifically bound probe
- Expose membrane to x-ray film
- Align exposed film with original plate
- Grow cells containing gene of interest in culture.
35Gene 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.
36Sequencing
- Sequencing determining the order and
arrangement of Gs, As, Ts and Cs in a segment
of DNA.
37Sequencing
- Lets review replication..
38Sequencing
- The Sanger sequencing method uses
dideoxy-nucleotides to generate all possible
fragments of the DNA molecule to be sequenced.
deoxynucleotide
dideoxynucleotide
39Sequencing
- Set up four different reactions
40Sequencing
- Load the four reactions in different wells of a
polyacrylamide gel to separate the fragments
41Sequencing
Sequencing Animation
42Human 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
43Human Genome Project
44Human Genome Project
- Consider ethical, legal and social issues
- Who owns your DNA?
45Human Genome Project
- Develop new lab technology
- Automated Sequencing
46Human Genome Project
- Disseminate genome information
- GenBank database
47Human Genome Project
- Analyze genetic variation among humans
- The genome is approximately 99.9 identical
between individuals of all nationalities and
backgrounds.
48Human Genome Project
- Map and sequence genomes of model organisms
- E.coli
- Arabidopsis thaliana
- Saccharomyces cerevisiae
- Drosophila melangaster
- Caenorhabditis elegans
- mus musculus
49PCR
- 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."
50PCR
- 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
51PCR
- Reactions are placed in a machine called a
thermal cycler. The machine cycles through three
temperatures.
52PCR
- Heat samples to 94C for a minute or so to
denature the double stranded template DNA.
53PCR
- Drop temperature to around 50 or 60C to allow
primers to anneal.
54PCR
- Maintain temperature at 72C for a minute or two
to allow the polymerase to elongate the new DNA
strands.
55PCR
- The thermal cycler repeats the denaturing,
annealing, and elongating temperatures
approximately 30 times.
PCR Animation
56PCR
- PCR amplification is logarithmic, meaning the
number of copies of the target is doubled every
cycle. (2n)
57PCR
PCR animation
58PCR
- 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
59Applications of Recombinant DNA Technology
60Chromosomal Location and Gene Copy Number
- Fluorescence in situ hybridization (FISH)
61Chromosomal Location and Gene Copy Number
- Southern Blot - molecular technique where DNA is
transferred onto a membrane from an agarose gel
and a probe is hybridized.
62Southern Blot
- The first step in preparing a Southern Blot is to
cut genomic DNA and run on an agarose gel.
63Southern Blot
- The next step is to blot or transfer single
stranded DNA fragments on to a nylon membrane.
64Southern Blot
- The next step is to hybridize a radioactively
labeled DNA probe to specific sequences on the
membrane.
65Southern 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..
66Southern Blot
Southern Blot Animation
67Studying 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
68Studying Gene Expression
- Real Time PCR (qPCR)
- Eliminates the need for running agarose gels
- Is quantitative
69Studying Gene Expression
Microarray animation