2. Fragmenting genomic DNA for cloning
Random methods are best
Mechanical shearing: sonication, nebulizer
Nuclease treatment (usually restriction digest): 4 base cutters, partial digest
Large fragments better than small, fewer clones to get coverage of large genome

3. Random fragmentation of genomic DNA:
Hydrodynamic shear (physical breakage)
-- sonication (vibrating metal probe)
-- nebulization (like asthma inhalers)
-- passage through small needle orifice
DNA must be repaired with DNA polymerase after these treatments
Enzymatic breakage
-- Restriction enzyme (4 cutter, partial digest)
CviJ (pyGCpy and puGCpu)
-- DNAse I (semi-random cleavage)

4. Early library construction
Partial digest
Size fractionate
Block EcoRI sites
Add linkers
Digest with EcoRI
Ligate to lambda
Package
Early library construction

5. Improved library construction
Partial digest: Sau3A (BamHI compatible ends
Phosphatase
Ligate to lambda
Package

6. Improved lambdas for libraries
More restriction sites
Sequences for phage RNA polymerase transcription (useful for probe synthesis)

7. But….
Cosmids
BACs
PACs
YACs
…can be used for cloning larger DNAs using similar methods… Why use lambda libraries?

8. Cosmids replicate as high copy number plasmids--tend to be unstable, deleting insert DNA (to reduce drag on cells)
BAC and YAC libraries difficult to prepare
larger-sized DNA more difficult to work with

9. Cloning cDNAs Prepared by reverse transcription of mRNA
Eukaryotic mRNAs--lack introns, often show variable splicing, cDNAs of these RNAs indicate how genes are actually expressed
Individual mRNA abundance varies widely: to isolate low abundance mRNAs by cDNA cloning, need to make libraries

10. Key points of cDNA cloning
mRNA source (tissue type) matters a lot
mRNA must be of high quality (no Rnases….)
Rare mRNAs can be enriched
e.g. “Subtractive cloning”
hybridize sample cDNA against immobilized RNA/cDNA from a “driver”, clone only those mRNAs that are not bound by the driver
This relies on differential mRNA expression between sample and “driver” mRNA populations

11. Gubler/Hoffman method (MC Chapter 11)
1) Synthesize first strand cDNA
2) Second strand cDNA
3) Methylate cDNA
4) Attach linkers or adaptors for cloning
5) Fractionate cDNA by size (select 2-8 kb)
6) Ligate cDNA into bacteriophage arms

12. cDNA libraries

14. cDNA synthesis
Make the first DNA strand from the mRNA template using reverse transcriptase
Remove the RNA
Make the second DNA strand from the first DNA strand

15. Primers for “first strand” cDNA synthesis
Oligo dT (binds polyA tails)
Oligo dT with adaptors (restriction sites)
Primers linked to a plasmid
Random primers

16. Random
priming

17. Second strand synthesis: early methods
Problem step
Loss of some of the mRNA 5’ end

18. Second strand synthesis--the Gubler/Hoffman protocol

19. Homopolymer
tailing

20. But many cDNAs are not full-length--how get only full-length cDNAs?
Utilize the 5’ CAP structure on eukaryotic mRNAs:

21. cDNA library construction using reverse transcriptase
cDNA Library Construction Kit (Clontech)

22. ESTs: Expressed Sequence Tags
Full length cDNAs hard to get, difficult to scale up
But short cDNA sequences are often useful
ID and map specific genes
“High throughput” allows very fast generation of bp sequences, or ESTs
Millions of ESTs in database
Useful in designing “microarrays” (later)

23. cDNA libraries: the easy way out
Pre-made cDNA libraries (organisms, tissues, variable conditions
Custom made cDNA libraries (you supply the mRNA)
“kits” for making your own cDNA library
(See Table 11-6 of Molecular Cloning for a directory)

24. Library construction DNA (entire genome…) Fragment the DNA
Clone in lambda phage vector
mRNA (only the expressed genes)
First strand cDNA
Second strand cDNA
Expressed sequence tags (ESTs)

25. Screening libraries for specific genes (finding the needle in the haystack)
Isolating individual clones
Screening by sequence
Hybridization
PCR
Screening by protein structure/biological function
Gene identification--diseases
Course reading #29

26. Overview of strategies for cloning genes

27. Improved library construction
You want to clone a gene from the human genome…
Partial digest: Sau3A (BamHI compatible ends)
Phosphatase
Ligate to lambda
Package
So you follow the protocol for
Or…buy a kit/premade library…

28. Basic “lytic” phage life cycle
100’s to 1000’s of plaques (individual phage infections)
Lawn of E. coli
But…which lambda clone (plaque) has the gene of interest????

29. How many recombinant DNA molecules are required in a library to get complete coverage of a genome?
p = probability of getting a specific piece of DNA
ln(1-p)
N =
ln(1-f)
f = fractional size of clone DNA relative to genome
N = number of clones needed

30. cDNA cloning: this calculation is harder…
ln(1 - p)
N =
ln(1 - f)
p = probability of getting a specific piece of DNA = 99%
f = fractional size of clone DNA relative to genome = base pairs (lambda capacity) / 3 x 10 9)
N = number of clones needed = 810,000
ln( )
N =
= 810,000
ln[1 - (1.7 x 104 / 3 x 109)]
cDNA cloning: this calculation is harder…

31. Screen by hybridization
Very fast
Applicable to a large number of clones
Can identify clones that are not full length
But you need to know at least some of the sequence of the gene you are after (more on this later)

32. Design of nucleic acid probes
Known sequences: eg. previously cloned cDNA to locate position in genome (identical match exists in library--stringent hybridization conditions)
Probes for non-identical but related sequences: finding a related gene in another species (non-identical match--reduce stringency of hybridization)
Probing for a gene from a sequenced protein: eg.
his-phe-pro-phe-met
4) Screen by PCR
make synthetic “mixed probe” (typically 16-mers)

33. “guessmers”: long, degenerate oligo probes
40-60 nts, alternative to short, “mixed probe”
Codon uncertainty mostly ignored
Most common codon used
Increased length improves specificity
Inosine substitutions at uncertain positions
Inosine pairs with all 4 bases
Low stringency hybridizations

34. “Colony hybridization” for ID of clones (like Southern blotting but without DNA isolation/gel electrophoresis)

35. Plaque-lift hybridization--using a lambda library
Can do this multiple times (replicate experiments)

37. Alternative to plating: arrayed libraries
Individual clones of library spotted onto membranes in high density arrays (tens of thousands of genes)
Membranes probed as described (a la microarrays)
Standardizable, centralizable

38. Using genomic DNA libraries for mapping: Chromosome “walking”
Prior to sequencing
It is possible to determine the order of clones in a contiguous sequence (contig)
Genes whose general location is known (by genetic mapping), but whose function is not known, can be found by starting with the genetic marker clone and “walking” away from it

39. Chromosome walking: how are individual clones in a genomic library positioned relative to each other?
The data
The genome “assembly”

40. Chromosome walking
Probing can be restricted to one direction with RNA probes generated from clone ends
Beware of “warping” to another chromosome because of repetitive sequence probes
Use YAC and BAC libraries to take larger steps

41. Improved lambdas for libraries
More restriction sites
Sequences for phage RNA polymerase transcription (useful for probe synthesis)

42. Expression libraries--alternative to hybridization
Gene product (protein) is made (by E. coli) and detected by variety of methods
Eukaryotic genes: cDNA library is essential (no introns, gene size small)
Screening:
Immunological
Functional

43. Immunological screening

44. The plaque lift: kind of like a Western blot
Detect antibody with secondary antibody conjugated to reporter enzyme for visualization

45. Functional cloning Genetic complementation:
Cloned DNA sequence corrects defect in host strain
Gain of function
Cloned DNA confers new function to host
Both of these require cloned DNA to be transcribed, translated into functional protein in host (eukaryotic protein in E. coli could cause problems)
And you need a good assay for expression!

46. Functional complementation: shaker gene
Shaker-2 mice have defects in the inner ear, poor balance, and deafness
The shaker 2 gene encodes myosin XV
Mutations in the human homolog can cause deafness

47. Subtractive cloning
Remove cDNAs that are common to two sources
Useful for isolation and detections of differentially expressed rare cDNAs
Example: differential expression from physiological change
“driver” DNA - immobilized
“test” cDNA (single stranded): labelled and then annealed to driver DNA
Remaining DNA has no counterpart in the driver cells--probe library to locate genes
Or use the remaining DNA to probe a microarray

48. Screening libraries for specific genes (finding the needle in the haystack)
Isolating individual clones
Screening by sequence
Hybridization
PCR
Screening by protein structure/biological function
Gene identification--diseases