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