Biotechnology

Genetic Research and Biotechnology

Restriction Enzymes

• These enzymes are isolated from bacteria and cut a DNA strand at a specific base pair sequence using a hydrolysis reaction.

• Depending on which restriction enzyme is used, the resulting ends may be one of two types:– 1. Sticky ends – ends of DNA fragments with single-

stranded overhangs.– 2. Blunt ends – ends of DNA fragments that are

perfectly paired.

Table of Restriction EnzymesMicroorganism of origin

Enzyme Recognition Site After restriction enzyme digestion

Echerichia coli EcoRI 5’-GAATTC-3’3’-CTTAAG-5’

5’-G AATTC-3’3’-CTTAA G-5’

Serratia marcescens SmaI 5’-GGGCCC-3’3’-CCCGGG-5’

5’-GGG CCC-3’3’-CCC GGG-5’

Streptomyces albus SalI 5’-GTCGAC-3’3’-CAGCTG-5’

5’-G TCGAC-3’3’-CAGCT G-5’

Plasmids

• Sometimes we want to excise a gene from a source DNA and express it in a different organism.

• This is accomplished through bacterial “machinery” know as plasmids.

• Plasmids are small, circular double-stranded DNA molecules.

• They exist in the bacterial cytoplasm but are not a part of the chromosome.

• They range in size from 1000-200 000 bp.

Plasmid

• Although plasmids are not part of the bacterial chromosome, they do offer benefits to the bacteria.

• They often carry genes for antibiotic resistance as well as resistance to toxic heavy metals and some herbicides.

How are they used in biotech?

• Restriction endonucleases are used to cut the plasmid in one area only so that it becomes linear.

• The foreign gene is cut with the same endonuclease and so, contains complementary ends.

• The foreign DNA is placed in solution with the linear plasmid.

• The foreign DNA fragment will anneal to the plasmid and permanently become a part of it with the help of DNA ligase forming the phosphodiester bonds.

• Now the plasmid is considered recombinant DNA and can be inserted into a bacterial cell.

• The cell will express the genes contained on the plasmid, including the foreign gene, making many copies.

• In this way, the gene will be cloned.

Transformation

• The process of introducing recombinant DNA into host bacterial cells is called transformation.

• The plasmid is called a vector because it can carry genes from one source to another.

• CaCl2 at 0 degrees C is used to stabilize the bacterial cell membrane.

• The solution of bacteria and plasmid is then subjected to a quick increase of heat which creates a draft that physically sweeps the plasmid into the cell.

• The cells are then returned to a comfortable temperature of 37 degrees C where they can then grow and reproduce.

• Not all cells will take up the plasmid so there must be a way to determine which ones contain the desired gene.

• Selective plating is used for this purpose.• The plasmid also contains a gene for antibiotic

resistance.

• If a bacteria takes up the plasmid, it will also be resistance to an antibiotic.

• If the nutrient agar on which the bacteria is being grown contains an antibiotic, then only those cells with resistance will grow. Others will die.

• In this way, we can select for the transformed bacteria.• To further ensure that the transformed bacteria has the desired

gene, it can be subjected to the original restriction enzyme, isolated and run on a gel to confirm the expected pattern of bands.

• Read page 293-294 to learn about how this process is used in medical biotech.

Plasmid Cloning

• http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html

Polymerase Chain Reaction (PCR)

• PCR is a more efficient and direct way of making copies of a DNA sequence without using plasmids.

• The process of PCR is similar to DNA replication.• 1. Strands of DNA are separated using heat (94-96

degrees C)• 2. DNA primers that are complementary to the

target sequence are added, one on each strand, but on opposite ends because the strands are anti-parallel.

• 3. Taq polymerase is a DNA polymerase isolated from a bacteria that lives in hot springs. This is used rather than DNA polymerase III to build the strands because the process takes place at 72 degrees C.

• Once the strands have been built, the cycle repeats itself, doubling the number of double-stranded copies of the target DNA.

• http://www.youtube.com/watch?v=ZmqqRPISg0g&feature=related

• http://www.youtube.com/watch?v=eEcy9k_KsDI&feature=related

• http://www.youtube.com/watch?v=x5yPkxCLads&feature=related

Textbook

• Reading: pages 286-291• Questions – page 291 - #1-6

DNA Fingerprinting or Profiling

• The DNA of every individual is unique.• This fact can be used to accurately identify an

individual based only on a sample of DNA which can be obtained from skin, hair, bodily fluids, etc.

• Biotechnology is used in this process.

The basic process

• DNA is a double helix composed of millions of nucleotide base pairs.

• The specific sequence of these base pairs is unique from one individual to the next.

• In order to identify the specific sequences, the DNA must be cut into smaller pieces.

• Restriction endonucleases (restriction enzymes) cut the DNA at a particular sequence.

• This creates DNA sections of different lengths.• Every individual will have a different

combination of segment lengths• Since DNA is so small, a process is needed to

visually see the different DNA segments.

• Gel Electrophoresis is used to visualize the unique pattern of a person’s DNA.

• The DNA sample is placed on a gel plate.• The gel allows the DNA to travel through from

one end to the other.• The different DNA segments separate on the

gel according to size; shorter segments travel farther.

• A DNA marker with known sizes of segments is run alongside the sample so the sample’s segment sizes can be estimated.

• No two people will have exactly the same pattern of DNA fragments on the gel except for identical twins.

• http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

Gel Electrophoresis

• This process separates DNA fragments based on size.• The differences in size exist because each fragment

(after being cut by a restriction enzyme) has a different number of base pairs.

• The DNA travels through the gel according to size.• The gel is porous and acts like a sieve with smaller

segments being able to navigate around the pores easier than larger segments.

• For this reason, smaller segments will travel farther than larger segments.

• The DNA travels through the gel because it is negatively charged because of it’s phosphate group which gives it a net -1 charge.

• An electrical current is run through the gel with the negative end closest to the samples and the positive end farthest away.

• The negative DNA is repelled by the closer negative end and travels toward the positive end.

• A DNA marker is loaded along with the sample and can be visualized as it migrates toward the other end.

• Once electrophoresis is complete, the gel is stained (used to be ethidium bromide but not so much anymore) so the sample can be seen.

• The pattern is then compared to the marker and the fragment sizes estimated.

• Each person’s pattern will be unique.

Gel Plate

Textbook

• Reading – pages 291-294• Questions – page 295: #7-10, 12

DNA Sequencing

• The Human Genome Project (HGP), initiated in 1990, had as one of it’s goals to determine the DNA sequence of all 3 billion base pairs of the human genome.

• It has since been completed but the process was very slow at first and it cost billions of dollars and numerous researchers.

• While the HGP used computer technology to read the sequence, this technology was made possible because of the lab techniques developed during the 70s and 80s.

• Since the completion of this project, sequencing has improved even more and is now more efficient and less costly.

Sanger Dideoxy Method

• http://www.youtube.com/watch?v=aPN8LP4YxPo&feature=related

• http://www.mwit.ac.th/~deardean/link/All%20Course/pic/secuencia.swf

Next-generation Automated sequencing

• Current methods are greatly improving the speed of sequencing.

• This is beneficial because medical science is working towards the use a person’s DNA sequence to diagnose and treat various diseases such as cancer.

• For example, a cancerous tumour can be sequenced to determine the exact nature of the mutation and determine a course of treatment.

Textbook

• Reading – pages 295-299• Review Questions – page 300: #1, 3, 4, 6, 7, 8,

9, 10, 11, 12.