This shortened week 2/17 Review Biotech ~Quiz tomorrow viruses and biotech Prokaryotes Friday and Monday

Ayoooooo Biotechnology Mr. Moegling Verdi e/ /126997/animation40.html

Shameless Podcast Plugs CRISPR Patient Zero

Biotechnology Biotechnology – the manipulation of organisms or their components to make useful products

Biotechnology 1.Recombinant Technology 2.Restriction Enzymes 3.PCR 4.Gel Electrophoresis 5.Gene Cloning 6.CRISPR 7.DNA Fingerprinting 8.ELISA 9.Microarrays 10.Nucleic Acid Hybridization

DNA Cloning Gene Cloning – The production of multiple copies of a single gene Often to clone a gene, a bacteria is used – The plasmid of the bacteria is more easily manipulated, and a selected portion of DNA (or a gene) is inserted into this plasmid – The bacteria then reproduce asexually, producing identical copies of their DNA

DNA Cloning – Each new cell now has the desired gene – This can also be used to produce large quantities of a desired protein (not just the DNA)

Cloning Vectors In gene cloning, the original plasmid is called a cloning vector A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there

Examples of Gene Cloning Genes for pest resistance has been inserted into plants Genes have been inserted into bacteria that allow them to break down oil spills HGH has been produced using gene cloning Proteins that have dissolve blood clots are produced using gene cloning

Restriction Enzymes Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites A restriction enzyme usually makes many cuts, yielding restriction fragments – EcoR1

Using Restriction Enzymes to Make Recombinant DNA The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments Animation: Restriction Enzymes Animation: Restriction Enzymes

Fig Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones

Fig Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2 One possible combination

Fig Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones One possible combination Recombinant DNA molecule DNA ligase seals strands. 3 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2

Cutting DNA The DNA plasmid is first isolated It is then cut by using restriction enzymes (Imagine below that this is an entire circular plasmid) G CTTAA CTTAA G CTTAA G CTTAA G

G CTTAA CTTAA G Sticky End The sticky ends allow outside pieces of DNA to be attached and inserted into the DNA

Transformation Transformation – the change of a bacteria cell due to the uptake in incorporation of foreign DNA Whenever a bacterial cell takes in new DNA it is said to have been ‘transformed’

Transforming Bacteria The gene for HGH is removed from a human cell

Plasmid The plasmid is cut at certain sequences in the DNA using the same Restriction Enzyme used to cut the HGH gene from a human cell The plasmid will have sticky ends in addition to the HGH gene

The gene for HGH is then mixed with the bacterial plasmid, and the HGH is incorporated into the plasmid DNA Ligase covalently bond the sugar phosphate backbone The plasmid is then added to the bacteria

The bacteria now has the gene for HGH, and has the ability to produce it This shows the process of transformation in bacteria

A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

Fig DNA in nucleus mRNAs in cytoplasm

Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA

Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA

Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA DNA polymerase

Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA DNA polymerase cDNA

Screening a Library for Clones Carrying a Gene of Interest A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene This process is called nucleic acid hybridization

Studying the Expression of Single Genes We can detect mRNA in a cell using nucleic acid hybridization, the base pairing of a strand of nucleic acid to its complementary sequence The complementary molecule in this case is a short single-stranded DNA or RNA called a nucleic acid probe Each probe is labeled with a fluorescent tag to allow visualization

The technique allows us to see the mRNA in place (in situ) in the intact organism and is thus called in situ hybridization

Figure  m

Another widely used method for comparing the amounts of specific mRNAs in several different samples is reverse transcriptase–polymerase chain reaction (RT-PCR) RT-PCR turns sample sets of mRNAs into double- stranded DNAs with the corresponding sequences

A probe can be synthesized that is complementary to the gene of interest For example, if the desired gene is – Then we would synthesize this probe G 5 3 …… GGCCCTTTAAA C 3 5 CCGGGAAATTT

The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest Once identified, the clone carrying the gene of interest can be cultured

Fig Probe DNA Radioactively labeled probe molecules Film Nylon membrane Multiwell plates holding library clones Location of DNA with the complementary sequence Gene of interest Single-stranded DNA from cell Nylon membrane TECHNIQUE

Expressing Cloned Eukaryotic Genes After a gene has been cloned, its protein product can be produced in larger amounts for research Cloned genes can be expressed as protein in either bacterial or eukaryotic cells

Gel Electrophoresis

Fig TECHNIQUE Nitrocellulose membrane (blot) Restriction fragments Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction fragments Gel electrophoresis I II III Radioactively labeled probe for  -globin gene DNA + restriction enzyme III Heterozygote II Sickle-cell allele I Normal  -globin allele Film over blot Probe detection Hybridization with radioactive probe Fragment from sickle-cell  -globin allele Fragment from normal  -globin allele Probe base-pairs with fragments Nitrocellulose blot

Fig a TECHNIQUE Nitrocellulose membrane (blot) Restriction fragments Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction fragmentsGel electrophoresis I II III DNA + restriction enzyme III Heterozygote II Sickle-cell allele I Normal  -globin allele 1 32

Fig b I II III Film over blot Probe detectionHybridization with radioactive probe Fragment from sickle-cell  -globin allele Fragment from normal  -globin allele Probe base-pairs with fragments Nitrocellulose blot 4 5 Radioactively labeled probe for  -globin gene

DNA Sequencing Relatively short DNA fragments can be sequenced by the dideoxy chain termination method Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment The DNA sequence can be read from the resulting spectrogram

Fig DNA (template strand) TECHNIQUE RESULTS DNA (template strand) DNA polymerase Primer Deoxyribonucleotides Shortest Dideoxyribonucleotides (fluorescently tagged) Labeled strands Longest Shortest labeled strand Longest labeled strand Laser Direction of movement of strands Detector Last base of longest labeled strand Last base of shortest labeled strand dATP dCTP dTTP dGTP ddATP ddCTP ddTTP ddGTP

Fig a DNA (template strand) TECHNIQUE DNA polymerase Primer DeoxyribonucleotidesDideoxyribonucleotides (fluorescently tagged) dATP dCTP dTTP dGTP ddATP ddCTP ddTTP ddGTP

Fig b TECHNIQUE RESULTS DNA (template strand) Shortest Labeled strands Longest Shortest labeled strand Longest labeled strand Laser Direction of movement of strands Detector Last base of longest labeled strand Last base of shortest labeled strand

The Polymerase Chain Reaction (PCR) The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules Goal is to Amplify DNA in Vitro

This bacteria, which Brock named Thermus aquaticus, thrives at just below the boiling point of water...

Researchers use this enzyme to make many copies of the DNA with a technique called polymerase chain reaction, or PCR.

In PCR, researchers will heat the DNA along with the Taq enzyme, along with a vast surplus of all the different nucleotides: G’s, C’s, A’s and T’s.

The strands will separate!(completely)

The mixture is then allowed to cool, and as it does the enzyme will attach the free nucleotides to the exposed strands..

.. leading to two copies of double-stranded DNA!

Fig Genomic DNA TECHNIQUE Cycle 1 yields 2 molecules Denaturation Annealing Extension Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Target sequence Primers New nucleo- tides

Fig. 20-8a 5 Genomic DNA TECHNIQUE Target sequence 3 3 5

Fig. 20-8b Cycle 1 yields 2 molecules Denaturation Annealing Extension Primers New nucleo- tides

Fig. 20-8c Cycle 2 yields 4 molecules

Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

CRISPR Clustered regularly-interspaced palindromic Repeats Immunity for bacteria against the enemy: virus How we use it In a Nutshell: Cut a DNA sequence at any given location

ELISA

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme.

the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample

Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measureable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.