1. Recombinant DNA TechnologyBY
NIKAM C.D.
ASSISTANT PROFESSOR,
DEPARTMENT OF MICROBIOLOGY,
S.M. JOSHI COLLEGE, HADAPSAR,PUNE.

2. Recombinant DNA Technology classic
isolate DNA,
cut DNA into workable sized fragments,
amplify the fragments for storage and subsequent analysis,
identify and isolate specific sequences,
characterize by size genomic location and sequence.

3. Cutting the DNA ~3,100,000,000 base pairs per human haploid genome,
average chromosome length is ~13 million base pairs,
DNA is cut with bacteria-derived enzymes,
The act of cutting is termed “digestion”.

4. Restriction Enzymes
…proteins that recognize specific, short nucleotide sequences and cut DNA at those sites,
…bacteria contain over 400 such enzymes and recognize and cut over 100 DNA sequences.

5. Palindromes stir grits no lemon, no melon
5’ G-A-A-T-T-C ’
3’ C-T-T-A-A-G ’

6. EcoRI
5’ G A-A-T-T-C ’
3’ C-T-T-A-A G ’

7. Sticky Ends
...single-stranded DNA overhangs resulting from restriction digestion.

8. EcoRI
5’ G A-A-T-T-C ’
3’ C-T-T-A-A G ’

9. RsaI Rhodopseuomonas sphaeroides
5’ G-T A-C ’
3’ C-A T-G ’

11. Restriction Fragment Length Sizes (predicted)
- IF -
25% A % T % G % C
and
Random Distribution of Nucleotides
i.e. p(specific base) = .25
- THEN -
Distance between cut sites is equal to 4n bases,
(n = number of base pairs in the recognition site)

12. Average Restriction Fragment Length
n = 4, 256 base pairs
n = 6, base pairs
n = 8, kb base pairs

14. Still Lots of Fragments
4 cutter: 3,000,000,000 bp
~256 bp = ~12,000,000 fragments
6 cutter: 3,000,000,000 bp
~4096 bp = ~700,000 fragments
8 cutter: 3,000,000,000 bp
~65.5 kb = ~46,000 fragments

15. Ligation
…sticky ends with complementary base pairs can form hydrogen bonds,
…DNA ligase: an enzyme that catalyzes the reformation of the phosphodiester bonds.

16. Ligation 5’-------G A-A-T-T-C--------3’ 3’-------C-T-T-A-A G--------5’
hydrogen bonds align,
DNA ligase covalently links

17. DNA is Profligate
…sticky ends with complementary base pairs can form hydrogen bonds,
…with DNA from any species.

18. Cloning Requires a Vector…
…specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study.
Requires a Vector…
...a specialized DNA sequence that can enter a living cell,
...signal its presence to an investigator,
... and provide a means of replication for itself and the foreign DNA it carries.

19. And, Vectors…
…contain unique restriction sites to facilitate the creation of recombinant DNA molecules,
... must also possess a distinguishing physical characteristic such as size or shape by which it can be purified away from the host cells genome.

20. Vectors Plasmid E. coli up to 15 kb, Phage E. coli up to 25 kb,
Cosmid E. coli up to 45 kb,
BAC E. coli kb,
YAC Yeast kb.

21. Step 1…restriction digests and ligation of fragments into cloning vectors,

22. Cloning Step 2 …vector-insert recombinants are inserted in host cells,
Transformation (via bacterial mechanisms);
plasmids
BACS
YACS
Transduction (via virus mechanisms);
phage
cosmids

23. Cloning Step 3 ...vectors contain selectable markers,
only cells that contain vector DNA will survive selection,
recombinant vectors can be discerned from empty vectors by additional markers.
Blue/White Cloning

24. Amplification
…if you were to grind me up with a giant mortar and pestle, and extract all of my DNA coding for a single gene, you’d get about 1 mg of DNA,
…two liters of bacterial culture carrying my gene in a recombinant DNA vector, would yield an equivalent mass of DNA.

25. Assignment
Read 10.1 and be prepared to clone a fragment of DNA using a plasmid as a vector.

26. PCR, Polymeras Chain Reaction
B-PCR vs A-PCR
PCR, Polymeras Chain Reaction

27. Today More DNA Science DNA Amplification II, Gel Electrophoresis,
Polymerase Chain Reaction,
6.7, pp ,
Gel Electrophoresis,
Southern Blots, Northern Blots,
6.6, pp ,
Clones and Libraries, pp

28. Polymerase Chain Reaction
Kary B. Mullis
Nobel Laureate, 1993
Chemistry
…invented by Kary Mullis while cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino,
"It was quiet and something just went, Click!"

29. "THE SUN HAD been hot that day in Mendocino County
"THE SUN HAD been hot that day in Mendocino County. A dry wind had come out of the east, and nobody knew how hot it had been until, around sunset, the wind stopped. I drove up from Berkeley through Cloverdale headed to Anderson Valley. The California buckeyes poked heavy blossoms out into Highway 128. The pink and white stalks hanging down into my headlights looked cold, but they were loaded with warmed oils that dominated the dimension of smell.
It seemed to be the night of the buckeyes, but something else was stirring.""My little silver Honda's front tires pulled us through the mountains. My hands felt the road and the turns. My mind drifted back to the lab. DNA chains coiled and floated. Lurid blue and pink images of electric molecules injected themselves somewhere between the mountain road and my eyes."
Opening words, Dancing Naked in the Mind Field, © 1998, by Dr. Kary Mullis, Pantheon Books.

30. Mullis…
... “PCR is a chemical procedure that will make the structures of the molecules of our genes as easy to see as billboards in the desert and as easy to manipulate as Tinkertoys”.
DNA

31. Making DNA: Components
Cell
PCR
ss DNA template
helicase, etc. ?
dNTPs
present
Primer
primase
DNA Polymerase
DNA
polIII
Environment
nucleus
test tube

32. Oligonucleotides specific primers
~ Odds of a Specific Sequence
20-mer: 9.1 x 10-13
...short pieces of synthetic DNA can be manufactured that contain any sequence,
…template specific!

33. Making One Strand Of DNA
add primer
Add Polymerase
Add dNTPs, etc.

34. Making Two More Strands
Must Denature
Separate Strands

35. Denaturing can’t use helicase in vitro
…DNA denaturing conditions such as high heat or low salt concentrations irreversibly denature or inactivate most polymerases,
…dNTPs are not affected by denaturation,
…primers are not affected by denaturation.

36. Making Two More Strands
add primer to second strand
Add polymerase, etc.

37. Denaturation Step Bad
…several rounds of in vitro replication could be performed (prior to PCR), however, accumulation of denatured polymerases quickly poisoned the reactions.

38. …bacteria discovered in a hot spring in Yellowstone Natural Park in 1965,
…lives in salty water that ranges from 70o - 75o C,
…thus, does DNA replication at very high temperatures.

39. Thermus aquaticus’ Enzymes
…basic research demonstrated that many enzymes isolated from Thermus aquaticus function at very high temperatures,
…temperatures nearing 100o C,
…DNA denaturating temperatures.

40. Click
…Kary Mullis realized that repetitive rounds of DNA synthesis could be performed by using a heat-stable polymerase,
… Thermus aquaticus: Taq polymerase.

42. Syllabus Update 6.1, 6.6 - 6.8, 10.1 for Friday,
read the rest of the chapter for review,
Master the Chapter 6 summary,
Master 6.8, 6.13, 6.18, 6.19, 10.6, 10.7, 10.9,
Read the Science Paper for Monday.

43. Genetics
…in the news.
…a hardcopy of the assigned paper (Monday) will earn you two of the 12.5 points on Wednesday’s quiz.

44. Options?
This?
Or This?
Seymour Binzer’s brilliant phage recombination experiment that revealed gene structure?
Development of the technology used to rapidly identify emerging virus?
A (H1:N1 info)

46. Exponential Synthesis
as few as 1 DNA templates required,
excess dNTPS,
excess primers,
multiple cycles.
Movie

47. Primers?
Entrez

48. PCR Applications …new applications are created every day,
PCR products can be used for mapping genes,
PCR products can be used as probes,
PCR products can be probed,
PCR can be used to identify genotypes,
PCR can be used to sequence DNA directly.

49. Molecular Probing Heterologous Hybridization
…genes (DNA), or gene products (RNA) can be identified based on hybridization to labeled molecules,
…DNA probes are short, single-stranded stretches of nucleic acid that are complementary to target nucleic acids,
s of base pairs in length,
…radioactive or fluorescent labeled for detection.

50. Probe
Add a “labelled” dNTP to an in vitro synthesis reaction.

51. Southern Blot

52. Northern Blot
mRNA is...
RNA

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

54. 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

55. Genomic Sequences and Coverage
N = ln( )
ln(1 - v/2,900,000,000)
v = average vector insert size
plasmid (5000 bp) = 2.7 x 106
phage (20 kb) = 6.7 x 105
BAC (125 kb) = 1.0 x 105
YAC (500 kb) = 27,000 clones

56. E. coli vs. Humans ln(1 - P) # Clones = ln(1 - v/g)
P = probability of including any one sequence.
v/g = insert size / genome size
E. coli
Genome = 4.6 mb
n = 4.6 mb / 20 kb insert = 230
P = 0.999
# Clones = 1585
Human
Genome = 2900 mb
n = 2900 mb / 20 kb insert = 145,000
P = 0.999
# Clones = 1,001,621

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

59. Reverse Transcriptase
1. RNA dependent, DNA synthesis.
2. RNA Degradation.
3. DNA dependent, DNA Synthesis.
Error Rate: 1 in 20,000 nucleotides.

60. mRNA
- polyadenylation.
- introns are spliced out.
-AAAA...

61. cDNA Construction in vitro

62. cDNA

63. Genomic vs cDNA ...Jeff’s gene, i.e. hemoglobin…
isolate red blood cells,
red blood cells make lots of hemoglobin, thus the mRNA is enriched for hemoglobin sequences,
construct a cDNA library,
isolation of hemoglobin clones is facilitated,
genomic: ~1 of 1,000,000 clones,
cDNA from red blood cells: 1 of 3 clones.

64. 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.

65. 1. Probe: cDNA, Target: Genomic

66. 2. Probe: Genomic, Target: cDNA

67. Assignment Study figure 6.27, pp 237,
Be able to describe the steps required to isolate a genomic clone using a cDNA clone as a molecular probe.

68. Cycle Sequencing PCR: 1 Strand Sequencing
PCR driven DNA sequence procedure,
non-exponential amplification,
Dideoxy sequencing method,
florescent indicators.

69. = Single Strand PCR dNTPs Template 1 Primer Taq Polymerase w/ Buffer
Cycles =
Polymerization until Taq falls off, linear amplification.

70. Cycle Sequencing Chain Termination
ddNTPs
dNTPs
Template
1 Primer
Taq Polymerase w/ Buffer
Cycles =
Polymerization until Taq hits ddNTP, Taq falls off.

71. Fluorescent ddNTPs
Plus: a preponderance of dNTPs

73. Cycle Sequencing Chain Termination
ddNTPs
dNTPs
Template
etc.
Another
Template
Lots of each sized fragment are produced, each with a specific florescent base on the end.base

74. Dosage Compensation …more Chapter 7
X chromosomes in females provide twice the genes, as in males,
Drosophila: female genes are expressed at 50% of the male levels,
Mammals: one X ho,olog in females is silenced.

75. Canadian Cat Scientists Sees it First
Barr Body

76. Lyon Hypothesis
Mary Lyon; in humans, X chromosomes from father and mother are randomly inactivated.

77. The structure of the chromosome is altered.
X Inactivation
Barr Body
The structure of the chromosome is altered.

78. X-Linked Mosaicism
Different cell lineages contribute to different body locations on the body.

79. Epigenesis
A change in gene regulation brought about without a change in DNA sequence,
often to the structure of the chromosome,
or through modification of the nucleotide bases,
or through post transcriptional regulation.

80. For Monday!
…READ IT! BRING YOUR COPY!