DNA Technologies Chapter 13

What is biotechnology? Biotechnology- is the use of organisms to perform practical tasks for humans – Analysis – Manipulation – Application DNA technology- manipulation and use of DNA at the molecular level

Recombinant DNA technology Recombines different molecules together Combination of genes (DNA) from different sources into one single molecule of DNA using a PLASMID: » CIRCULAR PIECE OF BACTERIAL DNA.

Important figures in DNA technology Joshua Lederberg and Edward Tatum – Discovered how bacteria transfer DNA between each other Bacteria creates a tunnel to connect and pass genetic information

How can DNA be inserted into a gene? 1.Through tunnel like connection mentioned earlier 2.Through a virus that carries pieces of bacterial DNA from one host to another. 3.Bacteria can take up loose pieces of DNA from surrounding environment We’ll touch upon these a little bit later

How can Recombinant DNA technology be useful?

The Genetically Modified Controversy Genetically Modified Organisms- (GMO) is any type of organism that has at least one artificially obtained gene. – GMO’s are either Transgenic- meaning that the gene is obtained from a different species Non-transgenic- gene from a different organism of the same species.

Engineering Beneficial Bacteria Bacteria can be useful in a variety of ways with DNA technologies – Contain Plasmids- circular bacterial DNA that is not part of the chromosomal DNA. Plasmids can be replicated in order to help with antibiotic resistance Can be used to create transgenic molecules to help create desired traits through gene cloning. Recombination animation

DNA cutting and pasting Restriction enzymes- enzymes that are used to cut DNA at a known location specific to each enzyme. – Cuts created what is known as “sticky ends” because they are not straight, which helps for rebuilding and insertion of DNA – Sticky ends are put back together using DNA ligase Sticky ends

Cut with restriction enzyme Ligase inserts new DNA into the plasmid Recombinant DNA Sticky ends created

Recombinant DNA Combining DNA from two or more organisms. 1. Restriction enzymes cut DNA 2. Sticky ends allow for new DNA to attach 3. DNA ligase attaches fragments

Recombinant DNA Restriction enzymes are used to cut DNA from desired source into multiple pieces. Same enzyme cuts the plasmid DNA in 2 locations in order to insert DNA at the sticky ends Recombinant plasmid taken up by the bacterial DNA Cell replication creating multiple copies of desired gene

Genomic Libraries Since restriction enzymes cut multiple locations on the desired DNA sequence sometimes different genes are cloned These genes are put into a genomic library- entire collection of cloned DNA from an organism - so they can be accessed later on

Nucleic Acid Probes Strands of DNA can be radioactively labeled in order to find a desired sequence along a given length of DNA – If ATTCATGGATC were the labeled probe then the known sequence of TAAGTACCTAG would be found located.

Prokaryotic genes Prokaryotes- singled celled organisms that lack a nuclei 3 important parts of gene regulation on prokaryotes – Promotor – Operator – repressor

RNA polymerase attaches to the promotor Operator = on/off switch If repressor is present and bound to operator then transcription is inhibited When more of a particular gene needs to be produced the repressor unbinds and allows for RNA polymerase to take action. When enough has been made then the repressor rebinds.

Eukaryotic regulation Similar to prokaryotic except transcription factors are used to regulate. TF are controlled by chemical signals in the body. – Example - insulin is needed only in the pancreas so only those cells produce the desired switch.

Other important terms Stem cells- undifferentiated cells that can divide to turn into any type of body cell. Important in biotechnology. Homeotic cells- cells that act as master controllers. – Direct the development of specific cells in specific locations

GMO’s continued Modification of crops – Elongates growing season – Pesticide resistance to insects and fungi – Tolerate different environmental conditions Modification of animals – Used to create vaccines for animals – Create larger more desirable traits – Lessen time of maturation – Better quality – Leaner muscles for meat

Cloning Very difficult to successfully create a cloned mammal. Nucleus of an animal cell is used to replace the nuclei of an unfertilized egg The new zygote is then implanted into the uterus of an animal If successful after gestation a new “cloned” animal will be born. Increase the production of animals. (mass produce)

Polymerase Chain Reaction PCR- is a type of biotechnology that uses a small piece of DNA to replicate into a larger amount of DNA. – requires ~1μg of DNA – Process of heating and cooling – Uses Primers- specific strands of DNA that pair with a known sequence to replicate based on base pairing

Steps of PCR Denaturation – Occurs at 94 o C which splits double stranded DNA into single strand which can then be replicated Annealing – Occurs at 54 o C which allows for DNA polymerase to attach and add new nucleotides to create another strand of DNA Extension – Takes place at 72 o C creates nucleotides available to be added to existing chain during upcoming cycles

PCR creates DNA in an exponential fasion

Gel Electrophoresis Compares different samples of DNA based separation due to charge and length 1. restriction enzymes- enzymes that recognize and cut DNA at specific sequences 2. Samples of DNA are placed into wells on an agarose gel. 3. Electrical charge put through gel to create current to move DNA fragments.

Electrophoresis Electrophoresis continued DNA has a negative charge and is attracted to the positive charge at the other end of the gel The smaller the DNA fragment the farther down the gel it will travel. Gel electrophoresis is a useful tool in identifying unknown samples of DNA to known samples

Shorter fragments travel farther down the gel toward the positive side Larger fragments stay closer to the top at the negatively charged region

How does Electrophoresis help? Genetic markers- pieces of DNA that vary amongst organisms of the same species – Genetic markers can be used to differentiate individuals and is the main factor for fingerprinting – Located within 97% of DNA, which is non-coding regions DNA fingerprinting- an individual's unique banding pattern on an electrophoresis gel, determined by restriction fragments of the person's – Using markers to identify an exact individual.