BIL 104 - Lecture 24

Introduction to Cloning and Biotechnology

What is a clone? It's a group of genetically identical molecules, cells, or organisms derived from a single ancestor.
A clone may be...

naturally produced from a single zygote (as in the case of human identical (=monozygotic) twins

natural vegetative reproduction of plants ("pupping" - e.g., eye of a potato)

artificially produced in the laboratory by one of several methods

Plant Cloning

Plants can be cloned from single, meristematic cells.

This is commonly done in agriculture.

Animal Cloning Animals can be cloned in several different ways...

embryo splitting

similar to producing monozygotic twins
a growing embryo is split into individual cells at an early stage, and each blastomere (embryo cell) is allowed to grow.

nuclear transfer

nucleus of recipient cell (unfertilized ovum) is destroyed (ablated)
nucleus of a donor cell (from a desired adult organism) is implanted
new cell is stimulated to divide as if it were a newly fertilized ovum
this is the method (first successfully used in 1986) that produced Dolly the sheep in 1997.
Dolly was the first cloned animal that overturned the previously held notion that embryonic cells could not be stimulated to return to early zygote condition, once the nucleus reaches a certain stage of specialization during development.

DNA Cloning (The Foundation of Recombinant DNA Technology) Clones need not be whole, living organisms. One can also clone DNA itself.

A large number of identical DNA molecules (or fragments of DNA molecules), each of which has an identical sequence, can be produced by cloning DNA molecules from a single, ancestral DNA molecule (or fragment).

In the early 1960's and 1970's, genetic and biochemical discoveries and techniques laid the foundation for later cloning of DNA molecules.

Collectively, these techniques are known as Recombinant DNA Technology

Some of the Goals of the DNA Technologist...

1. Isolation of a particular gene, part of a gene or region of a genome
2. Production of large quantities of a gene product (protein or RNA) for easier study of those molecules
3. Increased production efficiency for commercially made enzymes and drugs
4. Modification of existing organisms so that they express a particularly desirable trait not previously encoded in the genome.
5. Correction of genetic defects in complex organisms, including humans.

MAIN CONCEPTS and DEFINITIONS in RECOMBINANT DNA TECHNOLOGY: Making and Replicating a desired piece of DNA:

One little piece of DNA by itself can't be studied. You can't determine its base sequence or its products by looking at it under a microscope!

To effectively study DNA, one must manufacture a large quantity of a DNA segment of interest, "magnifying" it for easier study with biochemical methods.

To do this, recombinant DNA is made by splicing a DNA fragment of interest into a small DNA molecule (such as a bacterial plasmid) called a VECTOR.

Once this is done, one can make huge numbers of the desired DNA fragment by inserting the vector into a very busy piece of DNA in another live cell (such as a bacterium).

The bacterium "works" for you by allowing the vector to replicate. As the bacteria multiply, so does your desired DNA!

This magnified sample (your DNA clone) can then be extracted for further study.

A Few Definitions:

The organism from which the DNA of interest is extracted is called the DONOR.

The DNA into which the DNA of interest is inserted (often a bacterial plasmid) is called a VECTOR.

The organism (or DNA) into which the foreign DNA is inserted is called the RECIPIENT.

An organism containing an artificially inserted, foreign piece of DNA is said to be TRANSGENIC (i.e., the recipient becomes transgenic once the new DNA is inserted).

How is it Done?

To excise a piece of DNA from a donor organism, RESTRICTION ENZYMES are used. These act somewhat like "enzymatic scissors," slicing through the DNA at specific, recognized sequences.

Once the DNA is excised, DNA ligase is the "enzymatic glue" used to insert it into replicating DNA of the host cell.

Note that DNA ligase isn't picky: it can't tell the difference between foreign and host DNA (who'd figure it would ever have to?), and this enables the creation of hybrid DNA--DNA from two separate sources (sometimes different species!).

A vector molecule with an insert of foreign DNA is a RECOMBINANT DNA MOLECULE. DNA made from the combined DNA of two (or more) species is sometimes called CHIMERIC DNA after the beast of Greek Mythology. (Now why, do you suppose?)

Vectors are often mixed with bacterial strains which take them up and incorporate them into their own genomes, a process known as TRANSFORMATION)

Vectors may also be replicated autonomously (without being inserted into the bacterial DNA) as the bacterium goes about its daily business.

By growing the bacterial strain carrying the desired recombinant DNA vector, one can grow a large number of the desired DNA fragment. This is the DNA CLONE.

Once a large DNA clone (remember: a clone is a group of things, not a single individual!) has been grown, the researcher can

characterize the DNA (determine its base sequence)
make RNA from it
make protein from it (after you've made the RNA)
modify the DNA to see what happens when it mutates
reinsert it into a recipient organism for production of products or further study

Let's Take a Closer Look at Each of the Steps Above... Restriction Enzymes

First discovered in bacteria, RESTRICTION ENZYMES cut DNA at very specific DNA base sequences (called RESTRICTION SEQUENCES).

These enzymes are believed to be a bacterial defense against viruses.

Each Restriction Enzyme recognizes and cleaves a very specific sequence of DNA, like SO.

Restriction sequences are PALINDROMES: they read the same, forward and backward on the opposite strands.

Cutting with restriction enzymes creates highly reactive "sticky ends" that act as attachment points for other fragments of DNA with complementary restriction sequences.

By connecting pieces of DNA from two different species (that happen to have the same restriction sites), we create CHIMERIC DNA.

Note that restriction sites are a "happy accident" of nature. They have nothing to do with gene function in the organism in which they are found.
In fact, they are a defense mechanism, found primarily in bacteria, which function to fragment and destroy the DNA of invading bacteriophages (i.e, "bacterium-eating" viruses) before it can incorporate into the bacterial host's genome to do its dirty work.

Bacterial DNA is immune to the bacteria's own restriction enzymes: in its normal state a bacterium's own restriction sites are highly methylated (i.e., the bases have many methyl groups (-CH₃ attached), protecting them from the activity of the restriction enzymes.

Isn't evolution fantastic?

Restriction enzymes are named for the organism from which they were first isolated. For example

EcoRI is isolated from E. coli strain RY13.
Eco refers to the genus and species (1st letter of genus; 1st two letters of specific epithet)
R is the strain of E. coli
I (Roman numeral) indicates it was the first enzyme of that type isolated from E. coli RY13.
BamHI is isolated from Bacillus amyloliquefaciens strain H
Sau3A is isolated from Staphylococcus aureas strain 3A.
And so on.

Each enzyme recognizes and cuts specific DNA sequences. For example, BamHI recognizes the double stranded sequence:

5'--GGATCC--3'
3'--CCTAGG--5'

Here's how it works. Notice the "sticky" ends mentioned previously.

To summarize...

Most restriction enzymes cut only one specific restriction site
Restriction sites are recognized no matter what the DNA's species.
The number of cuts in an organism's DNA made by a particular restriction enzyme depends on the number of restriction sites (specific to that restriction enzyme) in that organism's DNA.
A fragment of DNA produced by a pair of adjacent cuts is called a RESTRICTION FRAGMENT.
A particular restriction enzyme will typically cut an organism's DNA in to many pieces, from several thousand to more than a million.
There is a great deal of variation in restriction sites, even within a species (Everyone in this room has different numbers and locations of restriction sites. Your restriction site numbers and locations are more similar to those of your close family members than to unrelated humans.
Although these DNA variations are not phenotypically expressed, the variants can be considered molecular "alleles," and they can be detected with sequencing techniques.
This is yet another type of genetic variation of interest to the evolutionary biologist.

Vectors

A VECTOR is a piece of DAN that carries a fragment of desired DNA into a living cell for replication.

Vectors can be any type of DNA that has an affinity for living cells:

plasmids (self-replicating, circular bits of DNA found in bacteria) can carry relatively small segments of desired DNA
Yeast Artificial Chromosome (YAC) - an artificially constructed "plasmid" that can carry large segments of DNA. It contains
- telomeres
- centromeres
- lots of restriction sequences for easy splicing
YAC can be spliced with many different types of genes/DNA fragments, inserted into live yeast, and then allowed to replicate as if it were a normal, natural yeast chromosome.
Because yeast reproduce very quickly, you can get tens of thousands of copies of your desired DNA fragments in relatively short time.
YAC has been used extensively in the Human Genome Project, to amplify segments of the human genome for easier study.
Viruses of various types may also be useful as vectors, since they have very specific affinities for specific tissues in the body.
- This means that specific viruses could be used to deliver genes for implantation into the cell's genome in specific tissues where those genes are needed, as in the case of gene therapy.
There are many types of viral vectors in use and under study for future use, including...
- retroviruses
- adenoviruses
- adeno-associated viruses
- herpes simplex virus
- rhinoviruses
- Human Immunodeficiency Virus (HIV)

Each has its benefits and drawbacks. The search for the perfect vector continues--because the perfect vector probably does not exist. (There's probably no single vector that will work for every purpose.

The overall object: get a vector that will allow you to clone large amounts of a desired DNA fragment by inserting it into a rapidly dividing cell. THE POLYMERASE CHAIN REACTION (PCR)

An alternative to cloning DNA fragments via insertion into vectors, and then introduction into bacterial or yeast hosts is the POLYMERASE CHAIN REACTION (PCR).
PCR allows rapid, efficient amplification of DNA sequences of interest.
Let us look at a nice little film...
PCR is probably the most widely used method for making large quantities of a desired fragment of DNA.

Once you have large quantities of a DNA Clone, what do you do with it? One of the most important aspects of DNA study is... DNA SEQUENCING

Therefore, the first important step is to determine the base sequence of your cloned fragment.

There are many different methods used for DNA sequencing, including

Dideoxy Method
Your assignment is to study the three diagrams in the link and understand the general workings of this protocol. Don't try to memorize it, but understand the basic ideas.