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 restrictionenzyme

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 DNA2. 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 transcriptionis 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 94oC which splits double stranded DNA into single strand which can then be replicated

• Annealing– Occurs at 54oC which allows for DNA polymerase to attach

and add new nucleotides to create another strand of DNA• Extension

– Takes place at 72oC 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 length1. restriction enzymes- enzymes that recognize and cut

DNA at specific sequences2. 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 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 topat 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.