Biotechnology

Biotechnology

• The manipulation of organisms or their genes for – Basic biological research– Medical diagnostics– Medical treatment (gene therapy)– Pharmaceutical production– Forensics– Environmental clean up– Agricultural applications– Genetic components to make useful products

Biotechnology• The genetic manipulation of organisms– Humans have been doing this for thousands of years

• Plant & animal breeding

Evolution & breeding of food plants

Evolution in morphology of Zea mays from ancestral teosinte (left) to modern corn (right). The middle figure shows possible hybrids of teosinte & early corn varieties

Evolution of breeding of food plants

• “descendants” of the wild mustard– Brassica spp.

Animal husbandry/breeding

Biotechnology today

• Genetic engineering– Manipulation of DNA• Recombinant DNA– Nucleotide sequences from two different sources

are combined in vitro into the same DNA molecule

Gene Cloning

• Preparing gene-sized pieces of DNA in identical copies– Materials needed: bacteria and their plasmids

• Used for making copies of a particular gene and producing a protein product

Gene cloning• Plasmid are small circular DNA molecules that replicate

separately from the bacterial chromosome– Carry extra genes– Can be exchanged– Naturally occur in bacteria

• Foreign DNA is inserted into a plasmid• Recombinant plasmid is inserted into a bacterial cell• Reproduction in the bacterial cell results in cloning of the

recombinant plasmid• Permits production of multiple copies of a single gene• The original plasmid is called a cloning vector

– DNA molecule that can carry foreign DNA into a host cell and replicate there

Fig. 20-2

DNA of chromosome

Cell containing geneof interest

Gene inserted intoplasmid

Plasmid put intobacterial cell

RecombinantDNA (plasmid)

Recombinantbacterium

Bacterialchromosome

Bacterium

Gene ofinterest

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Plasmid

Gene ofInterest

Protein expressedby gene of interest

Basic research andvarious applications

Copies of gene Protein harvested

Basicresearchon gene

Basicresearchon protein

Gene for pest resistance inserted into plants

Gene used to alter bacteria for cleaning up toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

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Transformation

• Bacteria are opportunists– Pick up foreign DNA wherever it may be hanging out

• Have surface transport proteins that are specialized for the uptake of naked DNA

– Import bits of chromosomes from other bacteria– Incorporate the DNA bits into their own chromosome

• Form of recombination

• While E. coli lacks this mechanism, it can be induced to take up small pieces of DNA if cultured in a medium with high calcium ions concentration

Swapping DNA• Genetic recombination by trading DNA

1 3 2

Cut DNA• Restriction enzymes– Cut DNA molecules at specific DNA sequences

• Restriction sites• Many cuts yielding restriction fragments= RFLP

(Restriction Fragment Length Polymorph)– Length depends upon the number of cuts made in a given

piece of DNA and location of each recognition sequence– If DNA is cut in a staggered way, fragments with “sticky

ends” that bond with complementary sticky ends of other fragments are produced• Symmetrical palindrome

– The sequence is the same when one strand is read left to right and the other strand is read right to left

Paste DNA

• Sticky ends allow – H bonds between complementary bases to anneal

• Ligase– Seals the bonds between restriction fragments– Joins sugar-phosphate bonds

Fig. 20-3-3Restriction site

DNA

Sticky end

Restriction enzymecuts sugar-phosphatebackbones.

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One possible combination

Recombinant DNA molecule

DNA ligaseseals strands.

3

DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.

2

Restriction enzymes• Discovered in the 1960s• Named according to specific system– Letters refer to organism from which enzyme is isolated

• First letter = genus• Next two letters = species• Fourth letter = strain• Roman numeral = whether isolated first, second, etc. • Ex. EcoRI= 1st restriction enzyme found in E.coli

Ex. Producing Clones of Cells Carrying Recombinant Plasmids

• Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid:– The hummingbird genomic DNA and a bacterial plasmid

are isolated– Both are digested with the same restriction enzyme• One that makes a single cut within the lacZ gene and

many cuts within the hummingbird DNA– (codes for enzyme that hydrolyses lactose and can also hydrolyze

a synthetic molecule to form a blue product)

Recombinant Clones• The fragments are mixed. – Some join by base pairing

• DNA ligase is added to bond the fragment sticky ends

• Some recombinant plasmids now contain hummingbird DNA

Recombinant clones• The DNA mixture is added

to bacteria that have been genetically engineered to accept it.– Bacteria have a mutation

in the lacZ gene making them unable to hydrolyze lactose or X-gal

• The bacteria are plated on agar containing ampicillin and X-gal which will select for the bacterial with recombinant plasmid

Recombinant Clones• Only cells that have the ampR gene will reproduce and

form colonies• Colonies with nonrecombinant plasmid will be blue

because they can hydrolyze X-gal• Colonies with recombinant plasmids, in which lacZ is

disrupted, will be white because they can’t hydrolyze X-gal• This results in the cloning of many hummingbird DNA

fragments, including the beta-globin gene

Storing Cloned Genes in DNA Libraries• A genomic library, made using bacteria, is the

collection of recombinant vector clones produced by cloning DNA fragments from an entire genome

• A genomic library, made using bacteriophages, is stored as a collection of phage clones

Each of these books is a clone of bacterial cells that contain copies of a particular foreign genome fragment in their recombinant plasmid

• A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert

• BACs are another type of vector used in DNA library construction

• Each clone occupies one well of this plastic plate

Storing Cloned Genes in DNA Libraries

• Complementary DNA (cDNA) library– 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 coding sequences of expressed genes

• Applications – If you want to clone a gene but don’t know what cell type expresses it– By making cDNA from cells of the same type at different times in the

life of an organism, changes in patterns of gene expression during development can be traced

Storing Cloned Genes in DNA Libraries

Making cDNA for a eukaryotic gene• Reverse transcriptase is added to a test tube

containing mRNA isolated from the cell

• Reverse transcriptase makes the first DNA strand using RNA as a template and a stretch of thymine deoxyribonucleotides as a DNA primer

Making cDNA for a eukaryotic gene

• mRNA is degraded by another enzyme

• DNA polymerase synthesizes the second strand using a primer in the reaction system

Making cDNA for a eukaryotic gene

• The result is cDNA which carries the complete coding sequence of the gene but not the introns

• Nucleic acid hybridization– A clone carrying the gene of interest can be identified with a nucleic acid

probe having a sequence complementary to the gene • 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

G5 3… …G GC C CT T TA A A

C3 5C CG G GA A AT T T

Screening a Library for Clones Carrying a Gene of Interest

DNA Probes

• Probe– Short, single stranded DNA molecule– Mix with denatured DNA

• DNA hybridization– Probe bonds to complementary DNA sequence

• Label – Probe is labeled for easy detection

• 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

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

Bacterial Expression Systems• Several technical difficulties hinder expression of

cloned eukaryotic genes in bacterial host cells• To overcome differences in promoters and other

DNA control sequences, scientists usually employ an expression vector– a cloning vector that contains a highly active prokaryotic

promoter

Eukaryotic Expression Systems• The use of cultured eukaryotic cells as host cells and yeast

artificial chromosomes (YACs) as vectors helps avoid gene expression problems– YACs behave normally in mitosis and can carry more DNA than a

plasmid• Eukaryotic hosts can provide the post-translational

modifications that many proteins require• Recombinant DNA can be introduced into eukaryotic cells by

– electroporation• applying a brief electrical pulse to create temporary holes in plasma

membranes– injecting DNA into cells using microscopically thin needles

• Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)

• The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA– Copying DNA without bacteria or plasmids– A three-step cycle—heating, cooling, and replication—

brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

PCR• Requires double-stranded DNA containing the

target sequence, a heat-resistant DNA polymerase, all four nucleotides, and two 15 to 20 DNA nucleotides that serve as primers– Primers define section of DNA to be cloned– One primer is complementary to one end of the target

sequence on one strand; the second primer is complementary to the other end of the sequence on the other strand

PCR1. Denaturation: Heat briefly

to separate strands of DNA2. Annealing: Cool to allow

primers to form hydrogen bonds with ends of target sequence

3. Extension: DNA polymerase adds nucleotides to the 3’end of each primer

PCR• After 3 cycles, ¼

of molecules match the target sequence exactly. After 30 cycles, almost 100% of the molecules match the target sequence

Kary Mullis- 1985• Development of PCR technique– A copying machine for DNA

DNA technology• DNA cloning allows researchers to – Compare genes and alleles between individuals– Locate gene expression in a body– Determine the role of a gene in an organism

• Several techniques are used to analyze the DNA of genes

DNA Fingerprinting – Uses

• Used to reveal a DNA pattern which is unique to an individual

• Crimework: rape and murder cases (forensics)• Paternity suits• Missing persons and unidentified bodies• Immigration disputes• Animal work - breeding

Gel Electrophoresis • Separation of DNA fragments by size using a molecular

sieve = agarose gel• DNA is negatively charged– Moves toward the positive electrode– Smaller fragments move faster and migrate further

• A DNA-binding dye (ex. Methylene blue stain) is added to make DNA bands visible

Measuring fragment size• Compare bands to known

“standard”– Usually lambda phage cut with

HindIII• Nice range of size with a

distinct pattern

• DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis

• useful for comparing two different DNA molecules, such as two alleles for a gene

• Useful to prepare pure samples of individual fragments

Restriction Fragment Analysis

Restriction Fragment Analysis

•Change in DNA sequence affects restriction enzyme “cut” site

•Will create different band pattern

• Combines gel electrophoresis of DNA fragments with nucleic acid hybridization– Specific DNA fragments can be identified using labeled

probes that hybridize to the DNA immobilized on a “blot” of gel

Southern Blotting

Southern Blotting1. Each DNA sample is mixed with the same restriction

enzyme. – Digestion of each sample yields a mixture of thousands of

restriction fragments

2. The restriction fragments are separated by electrophoresis

3. Capillary action pulls the alkaline solution upward through the gel, transferring the DNA to nitrocellulose membrane (blot); DNA is denatured in the process

Southern Blotting4. The nitrocellulose blot is exposed to a solution containing a radioactively labeled probe.• Probe molecules attach by base pairing to any restriction

fragments containing a part of the beta-globin gene

5. The radioactivity in the bound probe exposes the film to form an image corresponding to those bands containing DNA that base-paired with the probe

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

Dideoxynucleotides– “normal” N-bases– Dideoxy N-bases

• Missing O for bonding of next nucleotide

• Terminates chain• Radioactively

tagged

DNA Sequencing

1. Fragment to be sequenced is denatured into ss and incubated with ingredients necessary for DNA synthesis

DNA Sequencing2. Synthesis of each new strand starts at 3’ end of the primer and continues until a ddNTP is inserted at random instead of the normal nucleotide. • This prevents further elongation of the strand.• Set of labeled strands is generated with the color of the

tag representing the last nucleotide in the sequence

DNA Sequencing3. The labeled strands are separated by passage through a gel with shorter strands moving through faster. • A fluorescence detector senses the color of each tag as the strands

come through. • The color indicates the identity of the nucleotide at its end.• The sequence is complementary to the template strand and can be

read from bottom to top

Fred Sanger - 1978• This was his 2nd Nobel Prize!!– 1st was in 1958 for the protein

structure of insulin

Analyzing Gene Expression• Nucleic acid probes can hybridize with mRNAs

transcribed from a gene• Probes can be used to identify where or when a

gene is transcribed in an organism

Studying the Expression of Single Genes

• Changes in the expression of a gene during embryonic development can be tested using– Northern blotting

• combines gel electrophoresis of mRNA followed by hybridization with a probe on a membrane

• Identification of mRNA at a particular developmental stage suggests protein function at that stage

– Reverse transcriptase-polymerase chain reaction• is quicker and more sensitive• Reverse transcriptase is added to mRNA to make cDNA, which

serves as a template for PCR amplification of the gene of interest• The products are run on a gel and the mRNA of interest identified

Reverse transcriptase-polymerase chain reaction (RT-PCR)

1. Carried out by incubating mRNA with reverse transcriptase and other necessary components

2. Performed using primers specific to the particular gene

3. Will reveal amplified DNA products only in samples that contained mRNA transcribed from the beta-globin gene

4. mRNA for this gene is first expressed at stage 2

• In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism– This Drosophila embryo was incubated in a solution

containing probes for five different mRNAs. The embryo was then viewed using fluorescence microscopy. Each color marks where each gene is expressed as mRNA

Studying the Expression of Single Genes

Studying the Expression of Interacting Groups of Genes

• Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays– compare patterns of gene expression in different tissues, at

different times, or under different conditions– Before vs. after treatment– Cancer vs. normal cells– Wound healing vs. scarring– Stages of development

– Colors– Red = expression in 1 sample– Green = expression in other sample– Yellow = expression in both samples– Dark = low expression in both

Fig. 20-15TECHNIQUE

Isolate mRNA.

Make cDNA by reversetranscription, usingfluorescently labelednucleotides.

Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray.

Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.

Tissue sample

mRNA molecules

Labeled cDNA molecules(single strands)

DNA fragmentsrepresentingspecific genes

DNA microarraywith 2,400human genes

DNA microarray

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DNA Chip • Patented microarray technology

from Affymetrix– Automated DNA synthesis of genes

of interest on chip• Chips are more consistent• Smaller spots/more spots per chip

– Can buy specific chips• Human chip• Mouse chip• Etc.

Determining Gene Function• Disable the gene and observe the consequences• Using in vitro mutagenesis, mutations are introduced

into a cloned gene, altering or destroying its function– When the mutated gene is returned to the cell, the normal

gene’s function might be determined by examining the mutant’s phenotype

• Gene expression can also be silenced using RNA interference (RNAi)– Synthetic double-stranded RNA molecules matching the

sequence of a particular gene are used to break down or block the gene’s mRNA

Genomes• Complete genome sequences exist for a human,

chimpanzee, E. coli, brewer’s yeast, nematode, fruit fly, house mouse, rhesus macaque, and other organisms

• Comparisons of genomes among organisms provide information about the evolutionary history of genes and taxonomic groups

• Genomics – the study of whole sets of genes and their interactions

• Bioinformatics – the application of computational methods to the storage and

analysis of biological data– The Human Genome Project established databases and refined

analytical software to make data available on the Internet• This has accelerated progress in DNA sequence analysis

Centralized Resources for Analyzing Genome Sequences

• Bioinformatics resources are provided by a number of sources:– National Center for Biotechnology Information (NCBI)

• Created by the National Library of Medicine and the National Institutes of Health (NIH) European Molecular Biology Laboratory• Genbank, the NCBI database of sequences, doubles its data

approximately every 18 months– Online visitors can search for matches to

» A specific DNA sequence» A predicted protein sequence» Common stretches of amino acids in a protein

• The NCBI website also provides 3-D views of all protein structures that have been determined

– DNA Data Bank of Japan– European Molecular Biology Laboratory

Fig. 21-4

Identifying Protein-Coding Genes Within DNA Sequences

• Computer analysis of genome sequences helps identify sequences likely to encode proteins

• Comparison of sequences of “new” genes with those of known genes in other species may help identify new genes

Understanding Genes and Their Products at the Systems Level

• Proteomics is the systematic study of all proteins encoded by a genome– Proteins, not genes, carry out most of the activities of the

cell• A systems biology approach can be applied to define

gene circuits and protein interaction networks– Researchers working on Drosophila used powerful

computers and software to predict 4,700 protein products that participated in 4,000 interactions

– The systems biology approach is possible because of advances in bioinformatics

System Biology Approach to Protein Interactions• The global interaction map

shows a subset of the most statistically likely interactions among 2,300 proteins

• Colors correspond to the location of the protein in the cell– Green = nucleus– Blue = cytoplasm– Yellow = plasma membrane

Application of Systems Biology to Medicine

• A systems biology approach has several medical applications:– Cancer Genome Atlas project

• Monitors 2,000 genes in cancer cells for changes due to mutations and rearrangements

– Cancers and other diseases • Treatment can be individually tailored

following analysis of gene expression patterns in a patient

– In the future, DNA sequencing may highlight diseases to which an individual is predisposed

Human gene microarray chip

Genome Evolution

• Genomic evolution is based on mutations• The earliest forms of life likely had a minimal

number of genes, including only those necessary for survival and reproduction

• The size of genomes has increased over evolutionary time, with the extra genetic material providing raw material for gene diversification

Duplication of Entire Chromosome Sets

• Accidents in meiosis can lead to one or more extra sets of chromosomes, a condition known as polyploidy

• The genes in one or more of the extra sets can diverge by accumulating mutations; these variations may persist if the organism carrying them survives and reproduces

Alterations of Chromosome Structure

• Humans have 23 pairs of chromosomes, while chimpanzees have 24 pairs– Following the divergence of humans and

chimpanzees from a common ancestor, two ancestral chromosomes fused in the human line

• Duplications and inversions result from mistakes during meiotic recombination– have accelerated about 100 million years ago– This coincides with when large dinosaurs went

extinct and mammals diversified

• Comparative analysis between chromosomes of humans and 7 mammalian species paints a hypothetical chromosomal evolutionary history

• Chromosomal rearrangements are thought to contribute to the generation of new species

Duplication and Divergence of Gene-Sized Regions of DNA

• Unequal crossing over during prophase I of meiosis can result in one chromosome with a deletion and another with a duplication of a particular region

• Transposable elements (segment of DNA that can move within the genome) can provide sites for crossover between nonsister chromatids

Evolution of Genes with Related Functions: The Human Globin Genes

• Evolved from one common ancestral globin gene• Duplicated and diverged about 450–500 million

years ago• Mutations caused differences between the genes

in the globin family • Subsequent duplications of these genes and

random mutations gave rise to the present globin genes, which code for oxygen-binding proteins

• The similarity in the amino acid sequences of the various globin proteins supports this model of gene duplication and mutation

A model for the evolution of the human α-globin and β-globin gene families from a single ancestral globin gene

Evolution of Genes with Novel Functions

• The copies of some duplicated genes have diverged so much in evolution that the functions of their encoded proteins are now very different– For example the lysozyme gene was duplicated and

evolved into the α-lactalbumin gene in mammals but not in birds• Lysozyme is an enzyme that helps protect animals against

bacterial infection• α-lactalbumin is a nonenzymatic protein that plays a role in

milk production in mammals

Rearrangements of Parts of Genes: Exon Duplication and Exon Shuffling

• The duplication or repositioning of exons has contributed to genome evolution

• Errors in meiosis can result in an exon being duplicated on one chromosome and deleted from the homologous chromosome

• In exon shuffling, errors in meiotic recombination lead to some mixing and matching of exons, either within a gene or between two nonallelic genes

Fig. 21-14

Epidermal growthfactor gene with multipleEGF exons (green)

Fibronectin gene with multiple“finger” exons (orange)

Exonshuffling

Exonshuffling

Exonduplication

Plasminogen gene with a“kringle” exon (blue)

Portions of ancestral genes TPA gene as it exists todayTissue Plasminogen Activator

How Transposable Elements Contribute to Genome Evolution

• Multiple copies of similar transposable elements may facilitate recombination, or crossing over, between different chromosomes– Insertion of transposable elements within a protein-

coding sequence may block protein production– Insertion of transposable elements within a regulatory

sequence may increase or decrease protein production• Transposable elements may carry a gene or groups

of genes to a new location• Transposable elements may also create new sites

for alternative splicing in an RNA transcript