LECTURE                           RECOMBINANT DNA

In this lecture you will learn about:

Recombinant DNA means chunks of DNA can be cut and spliced into
 
1.  Exchange within the same species or between species
example: gene therapy
2.  From plants/animals to bacteria,  bacteria to plants/animals
example: human genes to bacteria to make useful product, also done with blood factors that control hemophilia
3.  From plants to animals, animals to plants

well they were talking about sticking a flounder gene for resistance to freezing into strawberries

4.  Constructed ..  Genes made by scientists

"Construction of Synthetic Genes for Analogs of Spider Silk Spidroin 1 and Their Expression in Tobacco Plants "

The purpose is to affect the production of proteins in some manner, either by
a.  change the gene fixing the defect (gene therapy)
b.  add a gene add the Bt gene for example to corn
c.  silence or inactivate a gene like targeting a gene that is out of control or makes a protein that gums up the works ("Methods and artificial genes for antagonizing the function of an oncogene")

 
Certain essential discoveries have made this possible:
1. DNA can be "melted" and will re-anneal into active DNA
2. DNA can be cut reproducibly by restriction enzymes

Enzymes were isolated from bacteria that cut DNA between very specific sequences of BASES, called RESTRICTION SITES. These fragments are also double stranded DNA. These enzymes are called ENDONUCLEASES, RESTRICTION ENDONUCLEASES OR RESTRICTION ENZYMES.

The sites cut by restriction enzymes are called "palindrome sequences". These sequences are like the word "Anna" or the phrase "Madam, I'm Adam". The word or phrase reads the same backwards or forwards. With DNA, the two complementary sides of the DNA read the same backwards and forwards. For example, the restriction enzyme Hind III makes "staggered" cuts between the two highlighted A's. (The "..." are unspecified stretches of DNA between the palindrome sequences).

3. DNA can be separated into specific lengths

DNA has a net negative electrical charge. In an electrical field where there are negative and positive electrodes, DNA will always move toward the positive electrode (your car battery has positive and negative electrodes or terminals). The agarose gel (very similar to Jello) has pores or holes filled with water. DNA is separated by length (or size) while it is being pulled through the pores of the gel toward the positive electrode. Smaller pieces of DNA move through the gel's pores more rapidly while larger pieces get stuck in the pores and move more slowly. Some DNA is so large that they never enter the gel. The DNA sample is always "loaded" into wells at the negative end of the gel. The path that DNA takes as it moves is called a "lane".

DNA is white when dry and colorless when wet. In order to see the DNA, a stain is used that fluoresces when illuminated with ultraviolet light. 


4. DNA can be sequenced and the DNA read and translated into proteins
5. DNA can be copied thousands of times- PCR

PCR is a way to amplify a specific sequence of DNA. Remember that DNA requires an RNA primer to get started. So SPECIFIC RNA primer is added and using the heat resistant DNA polymerase (found in bacteria that live in hot geysers) a polymerase chain reaction machine (called a thermal cycler) just heats the mix up to separate the DNA strands, cools it down to let the RNA hybridize and prime and the polymerase do its thing, then heat it up again, cool it down, etc.

In terms of DNA fingerprinting, this is the key to being able to use extremely small samples of DNA, like in a licked stamp, spit, chewing gum, cig butt, etc. 
 

6. Ability to cut and splice DNA into vectors
7. Vectors, like viruses of bacteria, plants and animals and plasmids that can passively be taken up by some bacterial species and used to move DNA into cells.  Some can be "shot" in, like those for papaya. Others can be injected in. 
8. Proteins can be sequenced and then DNA sequences identified, comparied even constructed and inserted into genomes. 

It is by this method that the collagen of dinosaurs were found to be most closely related to birds, specifically chickens. Click here



GLOSSARY:
ALLELES: variations of a gene.
BASES: Adenine (A), guanine (G), thymidine (T) and cytosine (C).
bp: base pairs: A bonded to T, G bonded to C.
CHROMOSOMES: one long continuous molecule of DNA. Humans have 46
CODES: contains the information needed to make a protein.
COMPLEMENTARY STRAND: a strand of DNA being copied from a template strand.
DNA REPLICATION: a DNA to DNA copy: xerox copy.
DNA BACKBONE: the continuous, rigid framework for the BASES.
DNA FINGERPRINTING: method which analyses the variation in DNA VNTR's and uses it to "match" with as a known suspects DNA pattern.
DOUBLE STRANDED DNA: two complementary chains of DNA bonded to each other.
FREQUENCY: the percentage of the population that has a particular allele, is determined by actual testing.
FRAGMENTS: double stranded pieces of DNA.
HOMOLOGOUS PAIRS: chromosomes that match. Humans have 23 pairs.
IN VITRO: experiments conducted outside of a living cell or organism.
Kb: Kilobase, 1000 base pairs.
NON-CODING DNA: DNA that does not code for proteins, often called "junk" DNA.
NUCLEOTIDES: the building blocks of DNA.
PALINDROME SEQUENCES: the word "Anna" or the phrase "Madam, I'm Adam". The word or phrase reads the same backwards r forwards.
PCR: polymerase chain reaction, the amplification of small quantities of DNA.
POLYMERASE: the enzyme that adds nucleotides onto the end of primers
POLYMORPHISM: differences.
PRIMER: a short, complementary sequence of nucleotides needed for copying DNA.
PROBABILITY: the percentage of the population that has the same two or more alleles, is calculated by multiplying frequencies of individual alleles.
PROBE: short, complementary copy of a section of the DNA of interest used to identify specific DNA.
RANDOM ASSORTMENT: process of reducing two sets of chromosomes into one set in eggs or sperm. Which copy of each chromosome ends up in a particular sperm or egg cell is random.
RESTRICTION SITES: palindromic DNA sequences where restriction enzymes cut.
RESTRICTION ENZYMES: enzymes that cut at restriction sites.
RESTRICTION FRAGMENTS: double stranded fragments of DNA cut with restriction enzymes.
SINGLE STRANDED DNA: DNA chain without a complementary bound chain
TANDEM REPEATS: areas of junk DNA where a sequences are repeated in a line.
TEMPLATE STRAND: the strand of DNA that is being copied.
UNZIPPED DNA: DNA that is single stranded.
VARIABLE NUMBER OF TANDEM REPEATS: VNTRs: regions of junk DNA that have a highly variable number of repeats in line in different people.

REVIEW QUESTIONS:
Discuss what each of these mean and how each of these are necessary to recombinant DNA.
1. DNA can be "melted" and will re-anneal into active DNA
2. DNA can be cut reproducibly by restriction enzymes
3. DNA can be separated into specific lengths and identified
4. DNA can be sequenced and read
5. Ability to cut and splice DNA into vectors
6. Vectors, like viruses of bacteria, plants and animals and plasmids that can passively be taken up by some bacterial species and used to move DNA into cells
7. The DNA-> protein code
8. PCR