1. Genomic and cDNA libraries

4. DNA Libraries …collections of cloned DNA fragments, genomic,
cDNA (coding sequences).

5. Genomic Library Construction
Cloning all fragments of an organisms genome = genomic library
Each fragment in a vector transformed into a bacterial cell = Book
The collection of all of the clones (thousands) is the genomic library
requirement: *random
* bigger is better

6. Genomic Library Construction
DNA fractioning
DNA shearing
to ensure that DNA fragments are of size suitable for cloning
Digest with enzyme with 4bp recognition site
One site every 256bp (1/4 x 1/4 x 1/4 x 1/4)
Do a partial digestion (not all sites cleaved)
Ligate fragments to vector
Transform into E. coli

7. Genomic Sequences and Coverage
N = ln(1 - P)
ln(1 - f)
N = number of clones
P = probability of recovering a sequence,
f = fraction of the genome of each clone
f = genome lenght
average lenght of insert

8. Results for the human genome
Probability Insert lenght
15kb kb
Results for the human genome

9. Making a genomic DNA library.

10. Production of overlapping restriction fragments by partial digestion of human genomic DNA with Sau3A. This restriction endonuclease recognizes the 4-bp sequence GATC and produces fragments with single-stranded sticky ends with this sequence on the 5 end of each strand. A hypothetical region of human genomic DNA showing the Sau3A recognition sites (red) is shown at the top. Partial digestion of this region of DNA would yield a variety of overlapping fragments (blue) ≈20 kb long. Use of such overlapping fragments increases the probability that all sequences in the genomic DNA will be represented in a l library.

11. Partial Digest

12. Cosmid Cloning

13. Cloning in Cosmids

15. Production of overlapping restriction fragments by partial digestion of human genomic DNA with Sau3A.

16. CHROMOSOME WALKING

17. CHROMOSOME WALKING

18. Chromosome ‘Walking’

19. DNA Libraries …collections of cloned DNA fragments, genomic,
cDNA (coding sequences).

20. Cloning euykaryotic genes in prkaryotes require special "tricks" because eukaryotic genes have introns which are removed in the nucleus of eukaryotic cells prior to translation
Introns can account for more than 90% of the length of a eukaryotic gene. It is hard to clone very long DNA segments. In addition, intron-containing eukaryotic genes cannot be expressed in a bacterial host because prokaryotes lack splicing apparatus.
To overcome these problems, instead of directly cloning a gene, one can clone cDNA, a DNA copy of gene mRNA.
An enzyme, reverse transcriptase, is used to produce cDNA

21. cDNA
…DNA synthesized from an mRNA template with the enzyme reverse transcriptase.

22. Reverse Transcriptase
1. RNA dependent, DNA synthesis.
2. RNA Degradation.
3. DNA dependent, DNA Synthesis.
Basically two types:
AMV (aviary myeloblastosis)
MMLV (Moloney Murine Leukemia Virus)
Error Rate: 1 in 20,000 nucleotides.

24. Isolation of mRNA by oligo-dT affinity chromatography.

25. cDNA Construction in vitro
RNA degradation by alkali
DNA pol and second strand synthesis
Cleavege of hairpin by Nuclease S1
Terminal transferase and ligation into vector

26. cDNA

27. Strategy to
synthetize
double strand cDNA

28. B) cDNA Synthesis mRNA primed mRNA mRNA/cDNA hybrid AAAAAn
Anneal oligo dT primer
primed mRNA
AAAAAn
TTTTT
Reverse Transcriptase
and dNTPs
mRNA/cDNA
hybrid
AAAAAn
TTTTT

29. mRNA/cDNA hybrid nicked RNA nicked RNA used as primers by Pol
Gubler Hoffman cDNA Synthesis
mRNA/cDNA
hybrid
AAAAAn
TTTTT
RNase H
AAAAAn
nicked RNA
TTTTT
DNA Pol I
nicked RNA used
as primers by Pol
AAAAA
TTTTT

30. cDNA Library 2nd strand cDNA in pieces ds cDNA
Gubler Hoffman cDNA Synthesis
2nd strand cDNA
in pieces
AAAAA
TTTTT
E. coli DNA
Ligase
AAAAA
ds cDNA
TTTTT
Clone into vector
cDNA Library

31. cDNA Libraries
…provide a ‘snap-shot’ of the genes expressed in a particular cell, at a particular time, or under specific condition,
…however, do not provide regulatory sequences.

32. cDNA Libraries Limits:
…using a oligo dT, library has 3’-rich sequences
Using random examers it’s possible overcome this drawbacks, but the average length of sequences is reduced.
…hard to isolate long and full-lenght transcripts.

33. CAPture method: AAA Full cDNA incomplete cDNA AAA AAA TTT TTT Rnase A
Chromatography for elF-4E
AAA
TTT
Elution of full-lenght cDNA

34. BAP= Alkaline phosphatase
TAP= acid pyrophosphatase

35. 5’ - RACE Rapid Amplification Complementary Ends
Sintesi primo filamento cDNA
AAAAA-3’
7-mG
Aggiunta coda omopolimera al primo filamento cDNA
Trasferasi terminale
+ dATP
Trascrittasi inversa
+ GSP1
GSP1 5’
3’-AAAAAAAAA
Sintesi secondo filamento cDNA
mRNA
TTTTT P
complementarietà al primer
P(dT)
TTTT
GSP1’
I° gruppo di amplificazioni 3’
AAAAA P’
II° gruppo di amplificazioni
GSP2
GSP2’
Prodotto PCR finale

36. 3’ - RACE Rapid Amplification Complementary Ends
Sintesi primo filamento cDNA
AAAAAAAA-3’
7-mG
Trascrittasi inversa
+ P(dT)
mRNA
TTTTT P
Sintesi secondo filamento cDNA
GSP1
complementarietà al primer
P(dT)
I° gruppo di amplificazioni 5’ 3’
AAAAA P’
GSP1’
GSP2
GSP2’
II° gruppo di amplificazioni
Prodotto PCR finale

40. Identification of a specific clone from a library by membrane hybridization to a radiolabeled probe

42. Source of the DNA Probe?
Probe DNA must have complementarity with target DNA
Two sources
Related organism - heterologous probe
Reverse translate protein sequence

43. DNA Probe Probe from Related Organism
sequences are related evolutionarily
not identical but similar enough
Heterologous Probe
Use yeast gene to isolate Human gene

44. DNA Probe Reverse translate protein sequence
Use knowledge of Genetic Code to obtain DNA sequence(s) based on protein seq.
MET = AUG in RNA
ATG in DNA (or 5’ CAT)

45. Genetic Code Table

46. Reverse Translation of Protein Seq.
MET-TRP-TYR-GLN-PHE-CYS-LYS-PRO
ATG-TGG-TAT-CAA-TTT-TGT-AGA-CCN
C G C C G
32 Different oligonucleotides for this peptide
sequence (due to degeneracy of code)

47. Radiolabeling of an oligonucleotide at the 5 and with phosphorus-32
Radiolabeling of an oligonucleotide at the 5 and with phosphorus-32. The three phosphate groups in ATP are designated the a, b, and g phosphates in order of their position away from the ribose ring of adenosine (Ad). ATP containing the radioactive isotope 32P in the g-phosphate position is called [g-32P]ATP. Kinase is the general term for enzymes that transfer the g-phosphate of ATP to specific substrates. Polynucleotide kinase can transfer the 32P-labeled g phosphate of [g-32P]ATP to the 5 end of a polynucleotide chain (either DNA or RNA). This reaction is commonly used to radiolabel synthetic oligonucleotides.

48. Labelling of PCR products using a radioactive
dNTP!

49. Designing oligonucleotide probes based on protein sequence
Designing oligonucleotide probes based on protein sequence. The determined sequences then are analyzed to identify the 6- or 7-aa region that can be encoded by the smallest number of possible DNA sequences. Because of the degeneracy of the genetic code, the 12-aa sequence (light green) shown here theoretically could be encoded by any of the DNA triplets below it, with the possible alternative bases at the same position indicated. For example, Phe-1 is encoded by TTT or TTC; Leu-2 is encoded by one of six possible triplets (CTT, CTC, CTA, CTG, TTA, or TTG). The region with the least degeneracy for a sequence of 20 bases (20-mer) is indicated by the red bracket. There are 48 possible DNA sequences in this 20-base region that could encode the peptide sequence Since the actual sequence of the gene is unknown, a degenerate 20-mer probe consisting of a mixture of all the possible 20-base oligonucleotides is prepared. If a cDNA or genomic library is screened with this degenerate probe, the one oligonucleotide that is perfectly complementary to the actual coding sequence (blue) will hybridize to it.

50. Colony PCR 2. Si mette su una reazione di PCR per ogni
clone che si vuole analizzare, risospendendo
nella mix di PCR una parte della colonia.Si
utilizza una coppia di primers specifica per
l’inserto clonato
1. Si piastra, come al solito, una trasformazione. I cloni trasformanti
possono contenere il solo vettore o il
vettore più l’inserto
M _
I cloni 1,2,3 e 4 contengono l’inserto. Il clone 4 contiene solo il vettore. “-” e “+” sono controlli negativo e positivo

51. Immunological Screening
Antibodies to the protein encoded by the desired gene can be used to screen a library

52. Immunological Screen 1 2 6 3 5 4 matrix cells protein transfer cells
master plate
matrix
cells
protein 1 2 6
transfer
cells
lyse cells
bind protein
subculture
cells from
master plate 3
Add 1°Ab;
wash
positive
signal 5 4
Add substrate
Add 2°Ab-Enzyme
wash

53. Immunological Screen of Library
detectable
product
substrate
reporter enzyme
2° Ab
1° Ab
target protein
matrix

55. o---- Cloning strategies ----o
Making DNA “libraries” (from genomic DNA, mRNA “transcriptome”)
Screening to identify a specific clone (the needle in the haystack)
-- by the sequence of the clone
-- by the structure or function of the expressed product of the clone
Course reading: #28 (and 29)

56. Overview of strategies for cloning genes
Get DNA
Ligate to vector
Transform or transfect
Look for the gene… 1) 2) 3) 4)

57. 1) Get DNA
Genomic DNA
RNA

58. Ligate to vector: how to make this reaction favorable?

59. There are lots of ways to identify a particular gene…
This yields a “library”, a representative set of all the pieces of DNA that make up a genome (or all the cDNAs that correspond to the “transcriptome”)
cDNAs from different tissues reflect the different RNA populations that you find in distinct cell types:
Hence “liver” vs. “brain” vs. “heart” cDNA libraries
There are lots of ways to identify a particular gene…

60. Overview of strategies for cloning genes