BiotechnologyATG AAT TCA GGC TAG GAT ATC CCA AGC TCG AGA ATA TCC AAG ATC TTG GCC ACC

TAC TTA AGT CCG ATC CTA TAG GGT TCG AGC TCT TAT ACC TTC TAC AAC CGG TGG

ATG AAT TCA GGC TAG GAT ATC CCA AGC TCG AGA ATA TCC AAG ATC TTG GCC ACC

TAC TTA AGT CCG ATC CTA TAG GGT TCG AGC TCT TAT ACC TTC TAC AAC CGG TGG

Intro activity: pg 277

A Brave New World

Genetic Engineering

• Biotechnology is mostly focused around the manipulation of DNA

• If you are going to engineer DNA and genes and organisms you need a set of tools to work with

• This is a survey of those tools…

• Single celled prokaryotes

• Reproduce by binary fission (mitosis)

• Rapid growth - new generation every 20 min

• Bacterial chromosome– Single circular chromosome

– Haploid

– Naked DNA (no histones)

– 4 million base pairs• 4300 genes

• 1/1000 of the DNA in a eukaryote

Bacteria Overview

Transformation

• Bacteria are opportunists

– Pick up naked foreign DNA wherever it may be

– Import bits of chromosomes from other bacteria

– Incorporated the DNA bits into their own chromosome

• Express new genes

• Transformation

• Form of recombination

Plasmids

• Small supplemental circles of DNA– 5000 to 20 000 base pairs– Self-replicating

• Carry extra genes– 2-30 genes– Genes for antibiotic resistance

• Can be exchanged between bacteria– Bacterial sex– Rapid evolution

• Can be imported from environment

How can plasmids help us?

• A way to get genes into bacteria easily

– Insert new gene into plasmid

– Insert plasmid into bacteria = vector

– Bacteria now expresses new gene

– Bacteria make new protein

Biotechnology • Plasmids used to insert new genes into bacteria

gene we want

cut DNA

cut plasmid DNA

insert “gene we want” into plasmid...“glue” together

ligase

like what?…insulin…HGH…lactase

Cut DNA?DNA scissors?

recombinant plasmid

How do we cut DNA?

• Restriction endonucleases or restriction enzymes

– Discovered in 1990s

– Evolved in bacteria to cut up foreign DNA

– “restrict” the action of attacking organism, by protecting against viruses and other bacteria

• Bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA

What do you notice about these phrases?

radar

racecar

Madam I’m Adam

Able was I ere I saw Elba

a man, a plan, a canal, Panama

Was it a bar or a bat I saw?

go hang a salami I’m a lasagna hog

palindromes

Restriction enzymes

• Action of enzyme

– cut DNA at specific sequences

• restriction site

– symmetrical “palindrome”

– produces protruding ends

• sticky ends

• will bind to any complementary DNA

• Many different enzymes

– named after organism they are found in

• EcoRI, HindIII, BamHI, SmaI

Madam I’m Adam

CTGAATTCCG

GACTTAAGGC

CTG|AATTCCG

GACTTAA|GGC

Discovery of restriction enzymes1960s | 1978

Werner Arber Daniel Nathans Hamilton O. Smith

Restriction enzymes are named for the organism they come from:EcoRI = 1st restriction enzyme found in E. coli

Restriction enzymes

• Cut DNA at specific sites

– leave “sticky ends”

GTAACG AATTCACGCTT

CATTGCTTAA GTGCGAA

GTAACGAATTCACGCTT

CATTGCTTAAGTGCGAA

restriction enzyme cut site

restriction enzyme cut site

Sticky ends• Cut other DNA with same enzymes

– leave “sticky ends” on both

– can glue DNA together at “sticky ends”

GTAACG AATTCACGCTT

CATTGCTTAA GTGCGAAgene you want

GGACCTG AATTCCGGATA

CCTGGACTTAA GGCCTAT

chromosome want to add gene to

GGACCTG AATTCACGCTT

CCTGGACTTAA GTGCGAAcombinedDNA

Sticky ends help glue genes togetherTTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTTAACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA

gene you want cut sitescut sites

AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTTTTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA

chromosome want to add gene tocut sites

AATTCTACGAATGGTTACATCGCCGGATGCTTACCAATGTAGCGGCTTAA isolated gene

sticky ends

chromosome with new gene added

TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATCCATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC

sticky ends stick together

DNA ligase joins the strands Recombinant DNA molecule

Biotechnology

• Recombinant DNA – means fragments of DNA composed of sequences originating from at least two different sources

• Restriction endonucleases – are also known as restriction enzymes – molecular scissors that cut double-stranded DNA at a specific base pair sequence

• Each R.E. recognizes a particular base sequence as its recognition site where it does the “cutting”

• Most recognition sites are four to eight base pairs long and palindromic

• http://www.dnalc.org/resources/animations/restriction.html

• http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

Methylases

• Restriction enzymes must be able to recognize the difference between foreign DNA and their own.

• Methylases are specific enzymes found in both prokaryotes and eukaryotes

• In prokaryotes they modify the recognition site of a respective restriction endonuclease by placing a methyl group on one of the bases, preventing the restriction DNA into fragments.

• These are important tools for molecular biologists when working with prokaryotic organisms, because they prevent a gene fragment from being cleaved in an undesired location.

DNA Ligase

• You’re already familiar with this enzyme from DNA replication.

• In biotech, it’s used to join sticky-ended fragments of DNA together, and reforms the phosphodiester bond.

• Sticky DNA fragments are easier to rejoin than blunt end fragments, which use T4 DNA ligase – an enzyme that comes from the T4 bacteriophage.

Gel Electrophoresis

• Once the desired gene has been excised from its source DNA, it must be separated from the remaining unwanted fragments.

• DNA fragments can be separated by gel electrophoresis – a process that works based on the properties of the DNA molecule

• Gel electrophoresis is like a “molecular sieve” –DNA fragments of various sizes (created by r.e.) will be separated from one another.

• The shorter a fragment is, the faster it will travel through the gel.

Here’s how gel’s work:

• A solution containing different sized fragments is placed in a well (depression) at the top of the gel

• The gel is a jelly-like square of buffer-containing electrolytes and agarose or polyacrylamide.

• DNA fragments are mixed with a loading dye (to make them visible) and glycerol (to weigh them down into the well)

• Using DC electricity, a negative charge is placed at the well end of the gel, and a positive charge is placed at the opposite end.

• The gel is covered in an electrolyte solution that conveys the current through the gel.

• The negatively charged DNA migrates to the opposite end (positive electrode), with the shorter fragments moving further down the gel.

• Once the DNA has moved apart, the fragments are visualized by staining them, producing a banding pattern.

• The size of the fragments can be measured by comparing with a molecular marker (a sample of fragments of known size run through the same gel)

• The fragments can also be removed and purified for further use.

• Gel electrophoresis can also be used to separate polypeptide fragments from protein molecules.

Plasmids

• Cutting out sections of DNA from an existing genome, and then annealing them to another DNA molecule is common practice in biotech.

• Bacteria often provide the necessary machinery to carry out this process.

• For example, the gene for insulin has been isolated and can now be expressed by bacterial cells, this is where most of the insulin used today comes from.

• The expression of genes is done through the insertion of DNA fragments in plasmids –small circular, double-stranded DNA molecules that exist within bacteria, independent of the bacterial cell.

• Plasmids often naturally contain genes for antibiotic resistance.

• Foreign genes are spliced into plasmids using restriction endonucleases.

Transformation

• As you will recall from Griffith’s experiments, the introduction of DNA from another source is known as transformation.

• Plasmids are used as vectors that carry desired genes into a host cell, creating transformed bacteria.

• Bacteria can be induced to accept plasmids by suspending them in a solution of calcium chloride (ionizes to Ca2+ and Cl-)at 0°C

• The bacterial membranes contain exposed negatively charged phosphates, which the positive calcium ions stabilize.

• The cold temperature freezes the membrane making it more rigid in structure.

• At this point, the entire solution is heat shocked to 42°C for approximately 90 seconds.

• The outside of the cell temperature being higher than the inside causes a draft that sweeps the plasmids into the bacterial cell through pores in the membrane.

• The bacteria are then incubated in a media suspension at 37°C.

How to isolate bacteria with the desired plasmid

• Selective plating allows transformed bacteria to be isolated from non-transformed bacteria.

• The vectors used for cloning carry an antibiotic-resistant gene – if bacteria were successfully transformed, they would be able to grow on media that contain the antibiotic, and the non-transformed bacteria would not survive the antibiotic.

Genetic Engineering

• The principles of genetic engineering were experimentally established in the early 1970s. Cohen and Boyer conducted a series of experiments that resulted in a method of selecting, recombining, and introducing new genes into bacteria via plasmid vectors.

• By 1978, genetic engineering had commercial applications, producing the first human hormone, somatostatin. These techniques are now used worldwide.

• Genetic engineering serves many purposes today, such as in the production of widely used and very essential drugs that improve the quality of life (i.e. insulin and somatropin, and in the agricultural world, such as in improvements like the reduction of frost in crops by spraying them with a genetically engineered bacteria that prevent ice from crystallizing.

PCR – Polymerase Chain Reaction

• PCR is a direct method of making copies of a desired section of DNA, using the same process as takes place during DNA replication.

• Instead of gyrase and DNA helicase – heat is used to separate the double strands of DNA from one another. (96° C – breaks H bonds)

• In PCR the RNA primers are replaced with DNA primers made in the laboratory.

• The DNA primers are complimentary to the area to be copied.

• The temperature is decrease to 65°C to allow the primers to anneal to the template DNA.

• Then Taq polymerase – an enzyme isolated from bacteria that live in hot springs builds complimentary strands using free nucleotides that are added to solution.

• The process of heating and cooling the DNA can be repeated many times to make many copies of the target DNA.

• PCR is used in forensic criminal investigations, medical diagnoses, evolutionary studies and genetic research.

RFLPs – Restriction Fragment Length Polymorphisms

• Any difference in DNA sequence that can be detected between individuals in called polymorphism.

• Polymorphisms occur between all individuals in both coding and non-coding regions of DNA.

• RFLP analysis involves the pattern of different lengths of DNA fragments produced by restriction enzyme digested and shown by complimentary radioactive probes.

RFLP process

• First the DNA is subjected to one or more r.e.s.

• The digested DNA is run on a gel, appearing as a long smear because the amount of DNA is so large and the bands so numerous that the fragments cannot be resolved.

• The gel is subjected to a chemical causing the double-stranded DNA to be denatured into ssDNA.

• Southern blotting procedure to transfer the DNA to a membrane is performed.

• The DNA is immersed in a radioactive solution of nucleotide probes, where they hybridize to the ssDNA.

Fig. 20-11a

TECHNIQUE

Nitrocellulosemembrane (blot)

Restrictionfragments

Alkalinesolution

DNA transfer (blotting)

Sponge

Gel

Heavyweight

Papertowels

Preparation of restriction fragments Gel electrophoresis

I II IIIDNA + restriction enzyme

III HeterozygoteII Sickle-cellallele

I Normal-globinallele

1 32

Fig. 20-11b

I II IIII II III

Film overblot

Probe detectionHybridization with radioactive probe

Fragment fromsickle-cell-globin allele

Fragment fromnormal -globin allele

Probe base-pairswith fragments

Nitrocellulose blot

4 5

Radioactively labeledprobe for -globin gene

DNA Sequencing – Sanger dideoxymethod

• A DNA template to be sequenced is treated to become single stranded.

• A short single-stranded radioactively labeled primer is added to one end of the DNA template.

• Identical copies of the primed ssDNA are placed in four reactions tubes, each containing DNA polymerase, free dNTPs

• In low concentrations each tube contains one of the four dideoxy analogues – a dNTP whose ribose sugar is missing the –OH group on its 3’ carbon.

• As nucleotides are incorporated into DNA, any time a ddNTP gets incorporated it terminates the chain.

• For example the sequence:3’ – AATGCATGCATTAGC – 5’

• In the tube containing dideoxy adenines, the following fragments will be created:

• 5’ – TTA – 3’• 5’ – TTACGTA – 3’• 5’ – TTACGTACGTA – 3’ • 5’ – TTACGTACGTAA – 3’• These fragments can be run on a gel, and can be

read to determine the DNA sequence.

Fig. 20-12b

TECHNIQUE

RESULTS

DNA (template strand)

Shortest

Labeled strands

Longest

Shortest labeled strand

Longest labeled strand

Laser

Directionof movementof strands

Detector

Last baseof longest

labeledstrand

Last baseof shortest

labeledstrand

Applications - Transgenic Plants

• Transgenic plants are those with introduced foreign genes using a Ti plasmid a vector found in soil bacteria that enters plant tissue through a wound and natural transforms it.

• Genes have been added to plants to produce greater yields, make hardier plants, make insect and virus resistant strains and make herbicide tolerant species.

• Since 1994, 40 genetically modified foods have been approved in Canada.

• Approximately 60% of all food found in groceries today contain food that has been genetically modified.

Applications

• Gene therapy

• Forensics