1. Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
The DNA fragments can be separated by size using gel electrophoresis.
Individual fragments can be identified and purified for further analysis or experimentation.

2. Figure 13.2 Separating Fragments of DNA by Gel Electrophoresis
Figure Separating Fragments of DNA by Gel Electrophoresis A mixture of DNA fragments is placed in a gel, and an electric field is applied across the gel. The negatively charged DNA moves toward the positive end of the field, with smaller molecules moving faster (and farther) than larger ones. After minutes to hours for separation, the electric power is shut off and the separated fragments can be analyzed.

3. Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
Information from electrophoresis :
Number—depends on number of times the restriction site occurs
Sizes—fragments of known sizes are included for comparison
Relative abundance—indicated by intensity of bands in the gel
After separation, gel regions can be cut out and the DNA purified and sequenced or used to make recombinant DNA.

4. Concept 13.1 Recombinant DNA Can Be Made in the Laboratory
DNA ligase catalyzes the joining of DNA fragments, such as Okazaki fragments, during replication.
With restriction enzymes to cut fragments and DNA ligase to combine them, recombinant DNA can be made.

5. Figure 13.3 Cutting and Joining DNA
Figure Cutting and Joining DNA Many restriction enzymes (EcoRI is shown here) make staggered cuts in DNA. EcoRI can be used to cut two different DNA molecules (blue and orange). The exposed bases can hydrogen-bond with complementary exposed bases on other DNA fragments, forming recombinant DNA. DNA ligase stabilizes the recombinant molecule by forming covalent phosphodiester bonds in the DNA backbone.
Two DNA fragments joined by ligase using “sticky ends’

6. Concept 13.2 DNA Can Genetically Transform Cells and Organisms
Recombinant DNA technology can be used to clone genes (make identical copies).
In transformation, genes are inserted into host bacterial or other cells. When the cell reproduces, it produces millions of cells, all with the cloned gene.
Called transfection if the host cells are from animals
The altered host cell is called transgenic.

7. Concept 13.2 DNA Can Genetically Transform Cells and Organisms
Research using model organisms:
Bacteria, especially E. coli; plasmids are easily manipulated
Yeasts (Saccharomyces), commonly used as eukaryotic hosts
Plant cells can be induced to dedifferentiate into unspecialized stem cells that can be transformed and then grown into plants with the recombinant DNA in all their cells.
Cultured animal cells are used to study expression of human or animal genes
Whole transgenic animals can also be created.

8. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
DNA fragments used for cloning come from a variety of sources.
The first step often involves creating a genomic library—a collection of DNA fragments that comprise the genome of an organism.
DNA fragments are inserted into host cells; transformed cells produce colonies on selective media, which can be probed using complementary DNA probes.

9. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
Smaller DNA libraries can be made from complementary DNA (cDNA).
mRNA is extracted from cells, then cDNA is produced by complementary base pairing, catalyzed by reverse transcriptase.
A cDNA library is a “snapshot” of the transcription pattern of the cell.
cDNA libraries are used to compare gene expression in different tissues at different stages of development.

10. Figure 13.7 Constructing Libraries
Figure Constructing Libraries Intact genomic DNA is too large to be introduced into host cells. (A) A genomic library can be made by breaking the DNA into small fragments, incorporating the fragments into a vector, and then transforming host cells with the recombinant vectors. Each colony of cells contains many copies of a small part of the genome. (B) Similarly, there are many mRNAs in a cell. These can be copied into cDNAs and a library made from them. The DNA in these colonies can then be isolated for analysis.

11. Cut up all of nuclear DNA from many cells of an organism
DNA libraries
Cut up all of nuclear DNA from many cells of an organism
restriction enzyme
Clone all fragments into many plasmids at same time
“shotgun” cloning
Create a stored collection of DNA fragments
petri dish has a collection of all DNA fragments from the organism

12. 2 1 3 4 engineered plasmid with selectable marker & screening system
Making a DNA library 2 1
engineered plasmid with selectable marker & screening system
all DNA from many cells of an organism is cut with restriction enzymes
gene of interest 3
all DNA fragments inserted into many plasmids
human genome = 3 billion bases
fragments are cut to ~5000 bases
therefore ~ 600,000 fragments per cell.
But you have to use many cells to make sure you have a complete set in the library, so… you may have millions of cells that you extracted the DNA from, so… you would need millions of colonies, so the human genome cloned into bacteria would be a walk-in freezer full of petri dishes. 4
clone plasmids into bacteria

13. But how do we find colony with our gene of interest in it?
DNA library
But how do we find colony with our gene of interest in it?
recombinant plasmids inserted into bacteria
gene of interest
human genome = 3 billion bases
fragments are cut to ~5000 bases
therefore ~ 600,000 fragments per cell.
But you have to use many cells to make sure you have a complete set in the library, so… you may have millions of cells that you extracted the DNA from, so… you would need millions of colonies, so the human genome cloned into bacteria would be a walk-in freezer full of petri dishes. ?
DNA Library plate of bacterial colonies storing & copying all genes from an organism (ex. human)

14. cDNA (complementary DNA) libraries
Collection of only the coding sequences of expressed genes
extract mRNA from cells
reverse transcriptase
RNA  DNA
from retroviruses
clone into plasmid
Applications
need edited DNA for expression in bacteria
human insulin
Could you imagine how much that first insulin clone was worth to Genentech? One little piece of DNA in a plasmid worth billions! It put them on the map & built a multi-billion dollar biotech company.

15. Concept 9.2 DNA Replicates Semiconservatively
Copies of DNA sequences can be made by the polymerase chain reaction (PCR) using:
A double-stranded DNA sample
Two primers complementary to the ends of the sequence to be copied
The four dNTPs
A DNA polymerase that works at high temperatures
Salts and a buffer to maintain pH

16. Concept 9.2 DNA Replicates Semiconservatively
PCR is a cyclic process in which a sequence of steps is repeated over and over again.
DNA replication is fast, so it takes only a short time to make millions of copies.
The sequences at each end of the amplified fragment must be known ahead of time, so that complementary primers can be made.
A pair of primers will usually bind to only a single region of DNA in an organism’s genome.

17. Figure 9.15 The Polymerase Chain Reaction
Figure The Polymerase Chain Reaction The steps in this cyclic process are repeated many times to produce millions of identical copies of a DNA fragment. This makes enough DNA for chemical analysis and genetic manipulations.

18. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
RT-PCR uses reverse transcriptase and PCR to create and amplify a specific cDNA sequence without making a library.
mRNA is isolated and cDNA is made, then a specific sequence is amplified by PCR.

19. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
Synthetic DNA:
Artificial synthesis of DNA is now fully automated.
Synthetic oligonucleotides used as primers in PCR can be customized.
Longer synthetic sequences can be pieced together to construct completely artificial genes.

20. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
Mutations in nature have been used to demonstrate cause-and-effect relationships.
Synthetic DNA can be manipulated to create specific mutations in order to study consequences of the mutation.
Example: The auxin response element in promoters of plant genes that are switched on in the presence of the hormone auxin

21. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
An artificial promoter containing many copies of the element was made and ligated to a reporter gene.
The recombinant DNA was used to transform Arabidopsis plants.
When the plants were treated with auxin, the reporter gene was switched on at very high levels.

22. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
Another way to determine a gene’s function is to inactivate it (“knockout” experiments):
In animals, these experiments often involve homologous recombination.
The normal allele is inserted into a plasmid and restriction enzymes are used to insert a reporter gene into the normal gene.
The extra DNA prevents functional mRNA from being made.

23. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
The recombinant plasmid is used to transfect mouse embryonic stem cells.
Stem cells—unspecialized cells that divide and differentiate into specialized cells
The gene sequence in the plasmid lines up with the homologous sequence on the mouse chromosome, and may be swapped by recombination.

24. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
The transfected stem cell is then transplanted into an early mouse embryo.
The resulting mouse is examined for consequences of carrying an inactivated gene.
Knockout mouse models have been developed for many diseases.

25. Figure 13.8 Making a Knockout Mouse
Figure Making a Knockout Mouse Animals carrying mutations are rare. Homologous recombination is used to replace a normal mouse gene with an inactivated copy of that gene, thus “knocking out” the gene. Discovering what happens to a mouse with an inactive gene tells us much about the normal role of that gene.

26. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
Another way to study gene function is to block translation.
RNA interference (RNAi): Translation of mRNA can be blocked by microRNAs and small interfering RNAs (siRNAs)
MicroRNAs and siRNAs are antisense RNA.
Antisense RNA can be synthesized and added to cells to prevent translation—the effects of the missing protein can then be determined.

27. Figure 13.9 Using siRNA to Block the Translation of mRNA
Figure Using siRNA to Block the Translation of mRNA (A) Normally an mRNA is translated to produce a protein. (B) Translation of a target mRNA can be prevented with a small interfering RNA (siRNA) that is complementary to part of the target mRNA.

28. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
DNA microarrays (“gene chips”) have a series of DNA sequences attached to a solid surface in an array of microscopic spots, each containing thousands of copies of a particular sequence.
Each oligonucleotide can hybridize with only one DNA or RNA sequence, and thus is a unique identifier of a gene.

29. Concept 13.3 Genes Come from Various Sources and Can Be Manipulated
Microarrays can be used to examine patterns of gene expression in different tissues under different conditions and to identify individual organisms with particular mutations.
Example: Women with a propensity for recurring breast cancer tumors have a gene expression signature that can be identified.

30. Figure 13.10 Using DNA Microarrays for Clinical Decision-Making
Figure Using DNA Microarrays for Clinical Decision-Making The pattern of expression of 70 genes in tumor tissues (the pattern of colored spots) indicates whether breast cancer is likely to recur. Actual arrays have more dots than shown here.

31. Create a slide with a sample of each gene from the organism
Microarrays
slide with spots of DNA
each spot = 1 gene
Create a slide with a sample of each gene from the organism
each spot is one gene
Convert mRNA  labeled cDNA
mRNA  cDNA
mRNA from cells
reverse transcriptase

32. Labeled cDNA hybridizes with DNA on slide
Microarrays
slide with spots of DNA
each spot = 1 gene
Labeled cDNA hybridizes with DNA on slide
each yellow spot = gene matched to mRNA
each yellow spot = expressed gene
cDNA matched to genomic DNA
mRNA  cDNA
Developed by Pat Brown at Stanford in late 1980s
Realized quickly he needed an automated system: robot spotter
Designed spotter & put plans on Internet for benefit of scientific community.

33. Application of Microarrays “DNA Chip”
2-color fluorescent tagging
Comparing treatments or conditions = Measuring change in gene expression
sick vs. healthy; cancer vs. normal cells
before vs. after treatment with drug
different stages in development
Color coding: label each condition with different color
red = gene expression in one sample
green = gene expression in other sample
yellow = gene expression in both samples
black = no or low expression in both
It’s all about comparisons!
Powerful research tool.