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CHAPTER 20DNA TECHNOLOGY AND
GENOMICS
DIABETES Insufficient insulin (gene product) in the body
Insulin in the seeds of this Safflower plant
Insulin in themilk of this Argentinian cow!
“Over hot pastrami and corned beef sandwiches, Herbert Boyer and Stanley Cohen opened the door to genetic engineering and laid the foundations for gene therapy and the biotechnology industry.”1996 Lemelson-MIT Prize Winners
DNA technology – using DNA as a tool for advancement in research and medicine
o Biotechnology – using “life” to make useful products
• Recombinant DNA -genes from two different sources - are combined in vitro into the same molecule.
• Genetic engineering - the direct manipulation of genes for practical purposes.
Insulin-a hormone produced by pancreas--a protein---a gene product
-Diabetes Treatments:-Supply artificial insulin-Then insulin (a gene product) has to be manufactured (how?)-Gene therapy to fix defect
Goals of Bio/DNA technology1) Isolate insulin or ‘gene of interest’ from the human genome
2) Engineer a recombinant DNA (ex. chimeric plasmid) that has the ‘gene of interest’
3) Clone the recombinant DNA (ex: in bacteria) to make million of copies
4) Now the gene of interest can be excised out of the cloned bacteria colonies using probes that bind selectively to the gene of interest (now you have millions of copies of your gene)
5) You can introduce this gene into an egg/sperm/balstocyst (early embryo) of any organism, or culture it to make your human gene’s protein
• Restriction Enzymes: found in bacteria naturally -used to cut foreign DNA in bacteria
• Most restrictions enzymes are very specific, recognize short DNA nucleotide sequences and cut at specific point in these sequences.– Bacteria protect their own
DNA by methylation.
Goal 1: Isolate gene of interest - ‘cut it’ out of human genomeGoal 2: Make recombinant DNA - ‘paste’ human gene in plasmid
• Restriction Enzymes:• Cut at Restriction Sites
on DNA• Restriction sites are
often palindromes• Restriction enzymes
leave sticky ends on the DNA they cut
• Paste - seal with DNA ligase
• How can you use this information to make recombinant DNA?
• Making Recombinant DNA
• Cut at SAME Restriction Sites on plasmid DNA and DONOR DNA (with gene of interest)
• Use DNA Ligase to join the sticky ends
• Step 1: Bacterial transformation - gets recombinant plasmid DNA into bacteria (use heat shock + CaCl2 to drill holes so foreign DNA is accepted)
• Now the recombinant DNA can be “cloned” this means - make many, many copies of the DNA by growing the bacteria on agar plates
Goal 3: Clone the recombinant DNA - reinsert the plasmid (vector) back into bacteria and let bacteria grow
Vector - used to clone the recombinant DNA
Step 2a: How will you know if the bacterial clone has the recombinant DNA?
Plasmid Vector has: Antibiotic (amp+/tet+) resistance gene - grow the bacterial culture in antibiotics and select colony that has resistanceRestriction sitesOri Site -starts replicationMarker/reporter that can be used to identify recombinant event (Ex. luciferase+ glowing gene from FIREFLY DNA)
Luc+ Amp+
Ori
LB = Only AgarNo Amp
LB + Amp LB+ Amp
No luc+ luc+No luc+
Amp Amp AmpLuc+
Negative Control Negative Control
Amp is an antibiotic; Amp+ is a gene on the plasmid that confers resistance. Luc+ makes bacteria glow.
Genes
Medium
AP Lab
Another Selection technique to pull out colony with gene of interest:Step 2b
-Nucleic acid hybridization depends on base pairing between DNA/RNA of interest and a complementary sequence, a nucleic acid probe
-A radioactive or fluorescent tag labels the probe.
- Bacterial colonies are transferred to a special filter paper (nitrocellulose)
- Colony containing the gene of interest can be identified using a probe that hybridizes (pairs) with the gene of interest
• Can you take a human gene and insert it into a bacteria using a plasmid vector and expect it to make protein?
• AHEM! What about introns? (remember a gene has both the introns and exons!)
• Solution: Use the spliced mRNA. Use reverse transcriptase and synthesize a complementary strand.– This is complementary DNA (cDNA), – Attach cDNA to a vector for replication,
transcription, and translation inside bacteria.
2 problems in making recombinant DNA in eukaryotes:
1) How do you get the correct mRNA out of a human cell (to make the cDNA)
In other words – how can the correct RNA/DNA be recognized in a cell?
2) How do you open up a eukaryotic cell and make it take up foreign DNA?
• Several techniques facilitate entry of foreign DNA in Eukaryotic Cells.– Electroporation- brief electrical pulses create a
temporary hole in the plasma membrane – Inject DNA into individual cells using microscopically thin
needles.– DNA is attached to microscopic metal particles and fired
into cells with a gun.
So, can you store all the human genes in a recombinant form?
• Book = bacteria• Each Chapter =
Different Plasmid• What will the
human gene be?• Passages in
chapters = human gene of interest
• Genomic Library – stores all the genes of an organisms in a library of vectors
• cDNA library - mRNAs are converted to complimentary or cDNA and stored in the library
• “Shotgun” cloning - a mixture of fragments from the entire genome is included in thousands of different recombinant plasmids.
Cloned genes are stored in DNA libraries
Celera- Shotgun approach:
Chop up the entire genome into small fragments
Determine the sequence of each individual part and then overlap segments to find the order of 300 billion base pairs!
Diploid Human DNA sequence determined in 2007 was Craig Venter’s!
Human genome project 1990 approach:
Use existing linkage maps and break up chromosomes into overlapping parts; make clones of the DNA -assigned to scienitists all over the country
Determine the sequence of each individual part and find the order of 300 billion base pairs!
• In addition to plasmids, bacteriophages can be cloning vectors for making libraries.
• The polymerase chain reaction (PCR) can clone DNA without using a vector
• HOW????
• WHAT DOES THIS IMPLY?
Do you always need a plasmid/vector to clone DNA?
• PCR-Reagents• 1) Primers - 20
nucleotide long single stranded DNA sequences that can hybridize with the gene of interest DNA at its very end.
• 2) Free nucleotides• 3) Special DNA
polymerase called Taq Polymerase - from bacteria that live in hot springs
Fig. 20.7
• PCR-Steps• 1) Heat to 95c - denature DNA
to break up ds DNA in gene of interest
• 2) Lower the temp. to 60c - Primers can bind to the ends of gene of interest
• 3) Next increase temp to 75c for Taq Polymerase to work well - it adds free nucleotides to 3’ end of primers
• 4) One cycle - 2 strands, 2 cycles - 4 copies, 3 cycles - 8 copies, …. 25 cycle - 33 million copies!
-Amplify a small amount of DNA quickly!
• Why PCR, when plasmids are available -
re-inventing the wheel, you say?• PCR can make billions of copies of a targeted
DNA segment in a few hours. • How can it do that?• PCR, is a three-step cycle: heating, cooling, and
replication• PCR, is like an atomic bomb explosion - HOW?• -a chain reaction that produces an exponentially
growing population of DNA molecules• Remember- PCR uses an UNUSUAL DNA
polymerase, isolated from bacteria living in hot springs, (can withstand the heat needed to separate the DNA strands at the start of each cycle).
Goal 4: Remove gene of interest from the cloned library using gel electrophoresis
• Comparison of 2 genomes: Gel Electrophoresis• DNA is –ve; Pass electric current• DNA will move to +ve electrode• So what?
Get DNA Fragments (use Restriction Enzyme digest)) Agarose
Gel
ParticlePores in gel thru which DNA fragments move
Know this! Rate of movement of DNA Fragment mainly depends on length or number of base pairs
Apply an electric current - DNA moves to +ve poleLong fragment
Short fragment
DNA Gel electrophoresis• Can detect differences in DNA
fragment size/length• DNA fragments arise from
restriction enzymes that cut DNA into different sizes
• Depending on where the ‘cutting’ or restriction sites lie on the noncoding regions of the chromosome you get different DNA gel patterns - so every person has a unique restriction digest profile - RFLP (rif- lips) Restriction Fragment Length Polymorphism (many forms)
• You inherit these restriction sites according to Mendelian rules
DNA FINGERPRINTING
DNA FINGERPRINTING USING RFLPS
• Restriction fragment analysis is sensitive enough to distinguish between two alleles of a gene that differ by only base pair in a restriction site.
Fig. 20.9
Remember these differences are in the noncoding parts - but restriction sites may lie close to a gene and serve as a ‘marker’ for that gene
• For our three individuals, the results of these steps show that individual III has a different restriction pattern than individuals I or II.
Fig. 20.10
Summary: Restriction fragment length polymorphisms (RFLPs) – we each have a different site where the restriction enzymes cut - DNA fingerprinting maps this out on a gel
• RFLPs can serve as a genetic marker for diseases
Southern blotting
• Helps identify the gene of interest in the gel electrophoresis bands
• Uses probes to hybridize with the gel band copy on a nitrocellulose paper
• The band of interest can be cut out and the gene of interest can be isolated - now we have millions of coies of pure gene of interest.
Insert the gene of interest in an egg/sperm and create a
transgenic animal
• Microinject the gene of interest into the egg/sperm nucleus!
• Animal makes human protein!• Plant is totipotent - so no need to use
embryos - get it into leaf or stem or root of a plant and culture it - these cells can make a transgenic embryo!
Microarrays: Attach all the dna/genes from an organism as single strands on a microarray plate. Ad cDNA from a patient .Spots where any of the cDNA hybridizes fluoresce with an intensity indicating the relative amount of the mRNA that was in the tissue.Gene chips
• In chromosome walking, the researcher starts with a known DNA segment (cloned, mapped, and sequenced) and “walks” along the DNA from that locus, producing a map of overlapping fragments.
Fig. 20.11
• Genomics, the study of genomes based on their DNA sequences, is yielding new insights into fundamental questions about genome organization, the control of gene expression, growth and development, and evolution.
• Rather than inferring genotype from phenotype like classical geneticists, molecular geneticists try to determine the impact on the phenotype of details of the genotype.
Genome sequences provide clues to important biological questions
• By doing more mixing and matching of modular elements, humans - and vertebrates in general - reach more complexity than flies or worms.
– The typical human gene probably specifies at least two or three different polypeptides by using different combinations of exons.
• Along with this is additional polypeptide diversity via post-translational processing.
– The human sequence suggests that our polypeptides tend to be more complicated than those of invertebrates.
• While humans do not seem to have more types of domains, the domains are put together in many more combinations.
• About half of the human genes were already known before the Human Genome Project.
• To determine what the others are and what they may do, scientists compare the sequences of new gene candidates with those of known genes.– In some cases, the sequence of a new gene candidate will be similar in
part with that of known gene, suggesting similar function.– In other cases, the new sequences will be similar to a sequence
encountered before, but of unknown function.– In still other cases, the sequence is entirely unlike anything ever seen
before.• About 30% of the E. coli genes are new to us.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Comparisons of genome sequences confirm very strongly the evolutionary connections between even distantly related organisms and the relevance of research on simpler organisms to our understanding of human biology.
– For example, yeast has a number of genes close enough to the human versions that they can substitute for them in a human cell.
– Researchers may determine what a human disease gene does by studying its normal counterpart in yeast.
– Bacterial sequences reveal unsuspected metabolic pathways that may have industrial or medical uses.
• Studies of genomes have also revealed how genes act together to produce a functioning organism through an unusually complex network of interactions among genes and their products.
• To determine which genes are transcribed under different situations, researchers isolate mRNA from particular cells and use the mRNA as templates to build a cDNA library.
• This cDNA can be compared to other collections of DNA by hybridization.– This will reveal which genes are active at different
developmental stages, in different tissues, or in tissues in different states of health.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Perhaps the most interesting genes discovered in genome sequencing and expression studies are those whose function is completely mysterious.
• One way to determine their function is to disable the gene and hope that the consequences provide clues to the gene’s normal function.– Using in vitro mutagenesis, specific changes
are introduced into a cloned gene, altering or destroying its function.
– When the mutated gene is returned to the cell, it may be possible to determine the function of the normal gene by examining the phenotype of the mutant.Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• In nonmammalian organisms, a simpler and faster method, RNA interference (RNAi), has been applied to silence the expression of selected genes.– This method uses synthetic double-stranded
RNA molecules matching the sequences of a particular gene to trigger breakdown of the gene’s mRNA.
– The mechanism underlying RNAi is still unknown.
– Scientists have only recently achieved some success in using the method to silence genes in mammalian cells.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• The next step after mapping and sequencing genomes is proteomics, the systematic study of full protein sets (proteomes) encoded by genomes.– One challenge is the sheer number of proteins in
humans and our close relatives because of alternative RNA splicing and post-translational modifications.
– Collecting all the proteins will be difficult because a cell’s proteins differ with cell type and its state.
– In addition, unlike DNA, proteins are extremely varied in structure and chemical and physical properties.
– Because proteins are the molecules that actually carry out cell activities, we must study them to learn how cells and organisms function.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Genomic and proteomics are giving biologists an increasingly global perspective on the study of life.
• Eric Lander and Robert Weinberg predict that complete catalogs of genes and proteins will change the discipline of biology dramatically.– “For the first time in a century, reductionists [are yielding]
ground to those trying to gain a holistic view of cells and tissues.”
• Advances in bioinformatics, the application of computer science and mathematics to genetic and other biological information, will play a crucial role in dealing with the enormous mass of data.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• These analyses will provide understanding of the spectrum of genetic variation in humans.– Because we are all probably descended from a small
population living in Africa 150,000 to 200,000 years ago, the amount of DNA variation in humans is small.
– Most of our diversity is in the form of single nucleotide polymorphisms (SNPs), single base-pair variations.• In humans, SNPs occur about once in 1,000 bases, meaning
that any two humans are 99.9% identical.
– The locations of the human SNP sites will provide useful markers for studying human evolution and for identifying disease genes and genes that influence our susceptibility to diseases, toxins or drugs.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
