Bio 423 Lecture 1

BB211: Cell and Molecular Biology Dr Eve Lutz Department of Bioscience

Recombinant DNA technology: Lecture 1 Introduction to Recombinant DNA technology Restriction enzymes and gel electrophoresis Background reading: Chapter 10 pp 205-206; Chapter 16 Klug, WS & Cummings, MR Essentials of Genetics, 4th ed. Please see the ReBase website for information regarding Restriction Enzymes: http://rebase.neb.com/rebase/rebhelp.html For Restriction Mapping, click on this link: Restriction Mapping Page For Additional Help, click on this link: Helpful Hints

An organism's genome contains virtually ALL the information necessary for its growth and development

Determining the molecular sequence of DNA that makes up the genome of different organisms is an international scientific goal, several laboratories are participating worldwide in this task (including the Wellcome Trust Sanger Institute and the Roslin Institute here in Britain). It is thought that having access to the complete DNA sequence of an organism can help us not only to decipher its biology but also help us understand major biological questions, for instance, what makes one species pathogenic whereas a related species is not. Or what are the genetic mechanisms which lead to disease and can they be reversed, halted or even prevented? It has the potential to help us understand very complex biological processes which are dependent on the interaction of a number of different genes (like development or the transmission and progression of particular diseases) and which have multifactoral causes and effects. And of course, in the day to day world, we use particular gene products (proteins and enzymes, peptides) that have been 'adapted' for commercial use (therapeutics, laundry detergents, genetically modified organisms such as tomatoes, rice, sheep etc etc).

How do we obtain DNA and how do we manipulate DNA? Quite straightforward to isolate DNA For instance, to isolate genomic DNA 1. Remove tissue from organism

2. Homogenize in lysis buffer containing guanidine thiocyanate 3. 4. 5. 6. 7. 8.

(denatures proteins) Mix with phenol/chloroform - removes proteins Keep aqueous phase (contains DNA) Add alcohol (ethanol or isopropanol) to precipitate DNA from solution Collect DNA pellet by centrifugation Dry DNA pellet and resuspend in buffer Store at 4°C

Each cell (with a few exceptions) carries a copy of the DNA sequences which make up the organism's genome. However, many genomes are large and complex (for instance the human genome is made up of ~3000 x 106 base pairs). A particular DNA sequence (for instance the allele of a gene) can be very small in comparison. And it probably occurs only once or twice within the genome (i.e. only one or two copies per cell). This means that a particular DNA sequence will be present as only a (very) small part within the complex mixture of DNA sequences that make up the genomic DNA of that organism. It is often necessary to 'break up' large DNA molecules into smaller, more manageable fragments - often to sizes ranging from 100 bp to 2 kb (bear in mind that each resulting DNA fragment is an individual molecule). These smaller fragments can then be manipulated more easily - to isolate particular DNA fragments, to characterize their molecular sequence, to determine their function, to determine their position in relation to other sequences within the genome, to use them to express proteins, etc . . .

How do we manipulate DNA? It used to be difficult to isolate enough of a particular DNA sequence to carry out further manipulation and/or characterization of its molecular sequence. DNA is a macromolecule - it is made up of a sequence of lots and lots of deoxyribonucleotides. Large DNA molecules can be fragmented using 'shearing' forces, in other words mechanical stress to 'shred it', thus creating smaller fragments. However, the resulting fragmentation is not reproducible - the breakage points can occur anywhere within the molecule, thus each DNA molecule will be randomly broken down and various different-sized fragments can be generated, any of which can have the DNA sequence of interest. A further difficulty in isolating a particular DNA fragment is that standard chemical/biochemical methods are not sufficient to distinguish any part of the genome from

another (after all one DNA molecule is chemically similar to another). Progress in understanding genetic mechanisms at the molecular level was slow. Then came the discovery of various bacterial and viral enzymes which modify and synthesize nucleic acids (DNA and RNA), along with the means to produce more outwith the organism from which they were originally isolated. The application of these enzymes for manipulating DNA (no matter what the source) led to the creation of Recombinant DNA Technology which has enabled great scientific advances in the field of biology, has created new scientific disciplines and has revolutionized our world.

Recombinant DNA Technology Techniques for - Isolation - Digestion - Fractionation - Purification of the TARGET fragment - Cloning into vectors - Transformation of host cell and selection - Replication - Analysis - Expression of DNA

DNA is manipulated using various enzymes that modify and/or synthesize it Until 1970 there were no convenient methods available for cutting DNA into discrete, manageable fragments. 1970 - The Beginning of the Revolution Discovery of a restriction enzyme in the bacterium Haemophilus influenzae

Restriction enzymes Enzymes that can cut (hydrolyse) DNA duplex at specific sites. Current DNA technology is totally dependent on restriction enzymes.

Restriction enzymes are endonucleases • • • • •

Bacterial enzymes Different bacterial strains express different restriction enzymes The names of restriction enzymes are derived from the name of the bacterial strain they are isolated from Cut (hydrolyse) DNA into defined and REPRODUCIBLE fragments Basic tools of gene cloning

Names of restriction endonucleases Titles of restriction enzymes are derived from the first letter of the genus + the first two letters of the species of organism from which they were isolated.

EcoRI - from Escherichia coli BamHI - from Bacillus amyloliquefaciens HindIII - from Haemophilus influenzae PstI - from Providencia stuartii Sau3AI - from Staphylococcus aureus AvaI - from Anabaena variabilis

Restriction enzymes recognize a specific short nucleotide sequence

This is known as a Restriction Site

The phosphodiester bond is cleaved between specific bases, one on each DNA strand

The product of each reaction is two double stranded DNA fragments

Restriction enzymes do not discriminate between DNA from different organisms Most restriction enzymes will cut DNA which contains their recognition sequence, no matter the source of the DNA

Restriction endonucleases are a natural part of the bacterial defense system • •

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Part of the restriction/modification system found in many bacteria These enzymes RESTRICT the ability of foreign DNA (such as bacteriophage DNA) to infect/invade the host bacterial cell by cutting it up (degrading it) The host DNA is MODIFIED by METHYLATION of the sequences these enzymes recognize o Methyl groups are added to C or A nucleotides in order to protect the bacterial host DNA from degradation by its own enzymes

Fig 7-5b, Lodish et al (4th ed)

Types of restriction enzymes •

Type I Recognise specific sequences·but then track along DNA (~1000-5000 bases) before cutting one of the strands

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and releasing a number of nucleotides (~75) where the cut is made. A second molecule of the endonuclease is required to cut the 2nd strand of the DNA o e.g. EcoK. 2+ o Require Mg , ATP and SAM (S-adenosyl methionine) cofactors for function Type II Recognise a specific target sequence in DNA, and then break the DNA (both strands), within or close to, the recognition site o e.g. EcoRI 2+ o Usually require Mg Type III Intermediate properties between type I and type II. Break both DNA strands at a defined distance from a recognition site o e.g. HgaI 2+ o Require Mg and ATP

Hundreds of restriction enzymes have been isolated and characterized • • •

Enables DNA to be cut into discrete, manageable fragments Type II enzymes are those used in the vast majority of molecular biology techniques Many are now commercially available

Each restriction enzyme will recognize its own particular site •

Some recognize very short sequences consisting of only 4 base pairs. These tend to cut DNA more frequently (generating smaller fragments) as the likelihood that any stretch of DNA sequence will contain these minimal recognition sites is high. i.e. approximately 1 site per 256 bases ([1/4]4)

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Some require longer recognition sequences (up to 8 bp). The longer the recognition sequence the less frequently these sites are likely to occur in any particular DNA sequence. Enzymes which cut DNA very infrequently are known as RARE cutters.

i.e. an 8 bp recognition site will occur approximately 1 per 65,536 bases ([1/4]8) The sites occur more randomly than predicted, so that digestion by any one enzyme will generate DNA fragments of different lengths

Some recognize more than one sequence • •

There are restriction enzymes which allow substitutions in one or more positions of their recognition sequences. Most common substitutions o purines (A or G), designated R o pyrimidines (C or T), designated Y o any nucleotide, designated N

For example HincII will allow two substitutions in each of two sites. It recognizes and cuts 4 different sequences. 5'-G T C GA C-3' 3'-C A G C T G-5'

5'-G T T G A C-3' 5'-G T C A A C-3' 5'-G T T A A C-3' 3'-C A A C T G-5' 3'-C A G T T G-5' 3'-C A A T T G-5'

The consensus HincII recognition site is designated 5'-G T Y R A C-3'

Many Type II restriction endonuclease recognize PALINDROMIC sequences

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Symmetrical sequences which read in the same order of nucleotide bases on each strand of DNA (always read 5' 3')

For example, EcoRI recognises the sequence 5'-G A A T T C-3' 3'-C T T A A G-5'

The high specificity for their recognition site means that DNA will be cut reproducibly into defined fragments • •

Generate restriction maps Isolate and clone specific DNA fragments

Different enzymes cut at different positions and can create single stranded ends ('sticky ends') •

Some generate 5' overhangs - eg: EcoRI

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Some generate 3' overhangs - eg: PstI

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Some generate blunt ends - eg: SmaI

Examples of restriction enzymes and the sequences they cleave Source microorganism

Enzyme

Arthrobacter luteus

Alu I

Recognition Site AG¬ CT

Ends produced Blunt

Bacillus amyloiquefaciens Bam HI H

G¬ GATCC

Sticky

Escherichia coli

Eco RI

G¬ AATTC

Sticky

Haemophilus gallinarum

Hga I

GACGC(N)5¬

Sticky

Haemophilus infulenzae

Hind III

A¬ AGCTT

Sticky

Providencia stuartii 164 Pst I

CTGCA¬ G

Sticky

Nocardia otitiscaviaruns Not I

GC¬ GGCCGC

Sticky

¬ GATC

Sticky

Staphylococcus aureus 3A

Sau 3A

Serratia marcesans

Sma I

CCC¬ GGG

Thermus aquaticus

Taq I

T¬ CGA

Blunt Sticky

The 'sticky' overhangs are known as COHESIVE ENDS •

The single stranded termini (or ends) can base pair (ANNEAL) with any complementary single stranded termini

This is the basis for RECOMBINANT DNA TECHNOLOGY •

Inserting foreign DNA into a cloning vector

Restriction enzymes are a useful tool for analyzing Recombinant DNA

After ligating a particular DNA sequence into a cloning vector, it is necessary to check that the correct fragment has been taken up. Sometimes it is also necessary to ensure that the foreign DNA sequence is in a certain orientation relative to sequences present in the cloning vector. • • •

Checking the size of the insert Checking the orientation of the insert Determining pattern of restriction sites within insert DNA

DNA fractionation

Separation of DNA fragments in order to isolate and analyze DNA cut by restriction enzymes Electrophoresis Linear DNA fragments of different sizes are resolved according to their size through gels made of polymeric materials such as polyacrylamide and agarose. For instance, agarose is a polysaccharide derived from seaweed - and gels formed from between 0.5% to 2% (mass/volume i.e. 0.5 to 2.0g agarose/100 ml of aqueous buffer) can be used to separate (resolve) most sizes of DNA DNA is electrophoresed through the agarose gel from the cathode (negative) to the anode (positive) when a voltage is applied, due to the net negative charge carried on DNA

When the DNA has been electrophoresed, the gel is stained in a solution containing the chemical ethidium bromide. This compound binds tightly to DNA (DNA chelator) and fluoresces strongly under UV light - allowing the visualization and detection of the DNA.

Other useful DNA modification enzymes used for manipulating DNA: Alkaline phosphatase

Removes phosphate groups from 5' ends of DNA (prevents unwanted re-ligation of cut DNA)

DNA ligase

Joins compatible ends of DNA fragments (blunt/blunt or complementary cohesive ends). Uses ATP

Synthesizes DNA complementary to a DNA template in DNA polymerase the 5'-to-3'direction. Starts from an oligonucleotide I primer with a 3' OH end Exonuclease III

Digests nucleotides progressively from a DNA strand in the 3' -to-5' direction

Polynucleotide kinase

Adds a phosphate group to the 5' end of double- or single-stranded DNA or RNA. Uses ATP

RNase A

Nuclease which digests RNA, not DNA

Taq DNA polymerase

Heat-stable DNA polymerase isolated from a thermostable microbe (Thermus aquaticus)