1
Basic techniques
--- Nucleic acid hybridizationcomplementary strands will associate and form double stranded molecules
--- Restriction EnzymesThese enzymes recognize and cleave DNA at specific sequences
--- BlottingAllows analysis of a single sequence in a mixture
--- DNA cloningThis allows the isolation and generation of a large number of copies of a given DNA sequence
--- TransformationStably integrating a piece of DNA into the genome of an organism
--- DNA sequencingDetermining the array of nucleotides in a DNA molecule
--- PCRamplification of known sequence
--- Genetic engineeringAltering the DNA sequence of a given piece of DNA
--- GenomicsAnalyzing changes in an entire genome
2
Nucleic acid hybridization
Complementary strands of DNA or RNA will specifically associate
DNA is heated to 100C, the hydrogen bonds linking the two strands are brokenThe double helix dissociates into single strands.
As the solution is allowed to cool, strands with complementary sequences readily re-form double helixes. This is called Nucleic acid hybridization.
5’ AAAAAAAATTTTAAAAAAA 3’
Will associate with
3’ TTTTTTTTAAAATTTTTTT 5’
This occurs with complementaryDNA/DNA, DNA/RNA, RNA/RNA
3
Li-Fraumeni syndrome
This technique is very sensitive and specific. A single 200 nucleotide sequence when added to a solution of a million sequences will specifically hybridize with the ONE complementary sequence
UsefulnessLi-Fraumeni syndrome
Individuals in a family have a propensity to develop tumors at an early age
Often these families have a deletion in the p53 gene
When this family has a child, they might want to know if their child has normal p53 or not
Nucleic acid hybridization provides a means to rapidly determine whether the sequence is present or not
Sequencing
4
Genomic DNA
Fragment DNA (clone)
Sequence fragments
Align fragments
Build consensus sequence
ACGCGATTCAACGCGATTCA GCGATTCAGGTTA GATTCAGGTTA CAGGTTACCACGC
ACGCGTAGCGC TAGCGCA
TAGCGCATTACAC
ACGCGATTCAGGTTACCACGCGTAGCGCATTACAC
Sequencing
5
Reference Genome- Number of donor DNAs are sequenced
Pieces of DNA are sequenced many times
Computers are used to overlap the pieces to generate contigs
Consensus sequence is reference genome
Sequences of individuals will vary from the reference genome
ACGCGATTCAGGTTACCACGCGTAGCGCATTACAC Reference Genome
ACGCGATTCAGGTTACCACGCGTAGCGCATTACAC MISTY
ACGCGGTTCAGGTTACCACGCGTAGCGCATTACAC NICK
ACGCGATTCAGGTTACCACGCGTAAAACATTACAC JESSE
ACGCGGTTCAGGTTACCCCGCGTAGCGCATTACAC DONNA
The sequence homology between Individuals is not perfect!!!
This allows us to assign a specific sequence to a specific Individual
6
Homology (molecular biology)
Regions of the DNA (gene or non-gene) that share similar nucleotide sequence
Sequence homology is a very important concept
Structural homology (nucleotide sequence) implies functional homology
Genes with a similar sequence are likely to function in a similar manner
Variation in sequence between individuals is also very Important
7
The method Isolate DNA
normal individual Patient
Fragment DNA, Heat to denature
Add radiolabeled ssDNA (p53 gene)(p53 probe)
Gradually and slowly cool solution
Radiolabeled p53 probe associates with DNA in normal individual
If patient is deficient for p53 gene Radiolabeled p53 probe is unable to associates with DNA in patient
Add enzyme (nuclease) that specifically degradesssDNA molecules. dsDNA remains degraded
Radiolabel present in dsDNA No radiolabel present in dsDNA(because p53 probe could not anneal)
Restriction Enzymes
8
What are Restriction enzymes
What are restriction enzyme sites in DNA
How do we map Restriction enzyme sites in DNA
How do we use restriction enzymes to clone pieces of DNA
How do we use restriction enzyme sites/maps to study individuals
9
Restriction Enzymes
Enzymes which Recognize a SPECIFIC DNA sequenceBIND that sequence andCUT The DNA at that specific sequence
SmaI is a Restriction enzyme
|5’ AAAACCCGGGAAAA3’3’ TTTTGGGCCCTTTT5’ |
This sequence is symmetrical. If one rotates it about the axisIt reads the same
EcoRI is another Restriction enzyme
|5’ AAAAGAATTCAAAA3’3’ TTTTCTTAAGTTTT5’ |
Some restriction enzymes recognize a specific sequence that is 4 bp longSome restriction enzymes recognize a specific sequence that is 6 bp longSome restriction enzymes recognize a specific sequence that is 8 bp long
Restriction enzyme digestion of DNA (linear genomic double stranded DNA)
OR
Restriction enzyme digestion of bacterial plasmid DNA (small double stranded circular DNA)
No digestion of RNA
No digestion of single stranded DNA
Restriction enzymes
BamHI
11
Linear/Circular DNA
No digestion of RNA
No digestion of single stranded DNA
A linear DNA molecule with ONE SmaI site will be cut into two fragments
A circular DNA molecule with ONE SmaI site will generate one DNA fragment
Blunt ends
Sticky ends
Blunt Vs Sticky
After digestion of DNA by a restriction enzyme the DNA ends are either blunt or sticky
14
Restriction sites
EcoRI is another commonly used restriction enzyme
Unlike SmaI which produces a blunt end, EcoRI produces sticky or cohesive ends (SINGLE STRANDED)
These cohesive ends facilitate formation of recombinant DNA molecules
SmaI- BLUNT ENDS
5’AAAAAAAAAAGGGGGGGGTTTTTTTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGGGGGTTTTTTTG AATTCAAAAAAAAGGGGGGGGTTTTTT3’3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAA GTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGGGGGTTTTTTTCCCGGGAAAAAAAAGGGGGGGGTTTTTT3’3’TTTTTTTTTTCCCCCCCCAAAAAAAGGGCCCTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGGGGGTTTTTTTCCC GGGAAAAAAAAGGGGGGGGTTTTTT3’3’TTTTTTTTTTCCCCCCCCAAAAAAAGGG CCCTTTTTTTTCCCCCCCCAAAAAA5’
15
5’AAAAAAAAAAGGGGGGGGTTTTTTTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGGGGGTTTTTTTG AATTCAAAAAAAAGGGGGGGGTTTTTT3’3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAA GTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGTTTTTTTGAATTCACGTACGTACGTACGTACGTACGTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’3’TTTTTTTTTACCCCAAAAAAACTTAAGTGCATGCATGCATGCATGCATGCACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGTTTTTTTG AATTCACGTACGTACGTACGTACGTACGTG AATTCAAAAAAAAGGGGGGGGTTTTTT3’3’TTTTTTTTTACCCCAAAAAAACTTAA GTGCATGCATGCATGCATGCATGCACTTAA GTTTTTTTTCCCCCCCCAAAAAA5’
Complementary sticky ends
AATTCAAAAAAAAGGGGGGGGTTT3’ GTTTTTTTTCCCCCCCCAAA5’
AAAAAAGGGGGGGGTTTTTTTGTTTTTTCCCCCCCCAAAAAAACTTAA
AATTCAAAAAAAAGGGGGGGGTTT3’ GTTTTTTTTCCCCCCCCAAA5’
AAAAAAGGGGGGGGTTTTTTTGTTTTTTCCCCCCCCAAAAAAACTTAA
AAAAAAGGGGGGGGTTTTTTTGTTTTTTCCCCCCCCAAAAAAACTTAA
GGCCCAAAAAAAAGGGGGGGGTTT3’ GTTTTTTTTCCCCCCCCAAA5’
SmaI AAACCCGGGAAA XmaI AAACCCGGGAAATTTGGGCCCTTT TTTGGGCCCTTT
EcoRI AAAGAATTCAAA MfeI AAACAATTGAAATTTCTTAAGTTT TTTGTTAACTTT
KpnI AAAGGTACCAAA Asp718 AAAGGTACCAAATTTCCATGGTTT TTTCCATGGTTT
Enzyme compatibility
19
Restriction maps
Restriction maps are descriptions of the number, type and distances between Restriction sites on a piece of DNA.Very useful for molecular biologists.Previously we used specific genes as markers on chromosome and Map units to indicate distance between the markers. Its like using specific landmarks to identify your location along a road. Restriction sites are also used as landmarks along a piece of DNA.
Restriction sites CAN serve as MARKERS ALONG the DNA. They can be used to generate a physical map of a specific DNA sequence can be created.
Hin
dII
I
EcoR
I
Sm
aI
PstI
205kb 300kb 4kb
pr vg cy11Mu 5Mu
NNNNNNNNNGAATTCNNNNNNNNNNNNAAGCTTNNNNNNNNNNNNCTGCAGNNNNNNNNNNCCCGGGNNNNNNNNNNNNNNNCTTAAGNNNNNNNNNNNNTTCGAANNNNNNNNNNNNGACGTCNNNNNNNNNNGGGCCCNNNNNN
AAAAAAAAAGAATTCTTTTTTTTTTTTAAGCTTCCCCCCCCCCCCCTGCAGGGGGGGGGGGCCCGGGAAAAAATTTTTTTTTCTTAAGAAAAAAAAAAAATTCGAAGGGGGGGGGGGGGACGTCCCCCCCCCCCGGGCCCTTTTTT
Pr Vg
Restriction maps
20
Human Genome is 1.5 billion basepairs long
There are 25,000 genes (markers)
Every gene is on average approximately 600,000 bp apart
EcoRI sites are on average 4000 bp apartHinDIII sites are on average 4000 bp apartEtc etc
There are many more Restriction enzyme sites (landmarks) on any one piece of DNA then there are genes (landmarks)
21
Sequence Divergence
The restriction map is a partial picture of the nucleotide sequence of a gene.By comparing restriction maps we can surmise differences in the sequence between species
Human
Chimp
Gibbon
NNNNNNNNGAATTCNNNNNNNNNNNNNNNNAAGCTTNNNNNNNNNNNNNNCTGCAGNNNNNNNNNNNNNNNNNNNNNNNCTTAAGNNNNNNNNNNNNNNNNTTCGAANNNNNNNNNNNNNNGACGTCNNNNNNNNNNNNNNN
GeneA
Mai California me rahta hu aur UCSC me padhata hu.
Mai California me rahta hu aur UCSC and UCLA me padhata hu.
Mai California me rahta hu aur mai sirf UCLA me padhta hu
22
Deletions and additionsEcoR
I
Hin
dII
I
EcoR
I
Hin
dII
I
EcoR
I
3 5 8 4
Normal Globin gene
Globin gene from a thallesimia patient
EcoR
I
Hin
dII
I
EcoR
I
Hin
dII
I
EcoR
I
3 5 3 4
With restriction maps, the relationship between a gene from two different individuals can be determined without having to actually sequence the gene from both individuals.
23
Very Large deletions or insertions can be studied using microscopy
Small to large deletions/insertions (100 bp to several kb) can be studied using restriction maps!!
Describing a DNA piece based on the pattern of restriction sites
Restriction map of a cloned piece of DNA
Linear or circular
S
K
E E
H
S
K
Restriction map
How do I do this for an unknown piece of DNA?
Analogy1
2
3
a
c
b
a
c
b
Goal: Identify the parts for this blob and describe the spatial relationship between the parts
Fit the parts together
Break the blob apart into its separate partsLook at the shapes and numbers of the parts
Restriction map of piece of DNA
Large amount of pure DNA (many copies of the same DNA is required) (Cloned)
Take (cloned) DNA in a tube,
Add restriction enzyme,
Allow enzyme to cut DNA at its binding sites
Run the digested DNA on a gel to resolve the DNA fragments based on size
Piece the fragments together to determine the linear order of the fragments (build the map)
Method
27
Gel electrophoresis
Mark
er
EcoR
IH
ind
III
EcoR
I/H
ind
III
-
+
EcoR
I
Hin
dII
I
EcoR
I
Hin
dII
I
EcoR
I
1 3 5 2
1
2
3
4
5
7
Mark
er
EcoR
I
Hin
dII
I
Agarose gel electrophoresis
The length of the DNA can be accurately determined byallowing the charged DNA torun through an agarose gel.
DNA is an anion (-ve
charged) and moves towards the Positive anode.
The rate of migration of aDNA fragment is inverselyproportional to its size.Larger the size, slower itsmovement.
EcoR
I/H
ind
III
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Mapping
You are given a 20 kb fragment of DNAAfter trying many enzymes you find that EcoRI and HindIII cut the fragment
HindIII 14kb and 6kb
EcoRI 12kb 6kb and 2kb
Solve the map
1
2
4
6
14
Mark
er
un
cu
t
Hin
dII
I
12
EcoR
I
20
614
6 14
6 14H
6212
E E
Or its mirror image
They are the same
What about EcoRI?
How do you arrange these three fragments wrt one another
29
Mapping
Since HindIII cut the 20kb fragment once, in which of the three EcoRI fragment does it cut?
A double digest with both enzymes will provide the answer
Fragments of 8kb, 6kb, 4kb and 2kb
The double digest does not alter the size of the 6kb and 2kb fragmentsThe 12kb fragment is lost. Also 8+4=12
8 4 6 2H E E1
2
4
6
14
Mark
er
EcoR
IH
ind
III
12
EcoR
I+H
ind
III
8
412 6 2
Partial Map only!!!! Not enough information
30
New Mapping
How are these fragments ordered?
The HindIII single digest tells us that they must be ordered so that One side adds up to 6kb and the other side adds up to 14kb
1
2
4
6
14
Mark
er
EcoR
IH
ind
III
12
EcoR
I+
Hin
dII
I
32
Mapping
HindIII EcoRI HindIII/EcoRI14 12 86 6 6
42 2
6 2 12
4 8HindIII
12 & 8
6 2 12
4816 & 4
6 212
4 810 & 10
6 212
4814 & 6
62 12
4 812 & 8
62 12
4816 & 4
34
Different Mapping example
Hi Ec Hi/Ec12 12 88 6 6
42 2
Ps Ps/Ec13 127 5
21
Three different enzymesHiEcPs
36
Mapping
HindIII EcoRI HindIII/EcoRI12 12 88 6 6
42 2
HindIII
12 & 8
12 & 8
6 2 12
4 8
12
62 12
4 812
6 2 12
8 4
16
16 & 4
H
H
H
E E
E E
E E
38
Mapping
6 2 12
EcoRI PstI PstI/EcoRI12 13 126 7 52 2
1
5 1Pst I
5 & 15
6 2 12
511 & 19
62 12
62 12
5 1
7 & 13
62 12
51
3 & 17
4 8
E E
E E
P
P
E E
E E
E E
P
P
H
40
Mapping deletions
Say you isolated this DNA from a region coding for GeneA, from a normal Patient and one suffering from a syndrome.
The fragment was 17kb in the affected individual rather than 20kb in the normal patient
6 8 2E H E
There is a 3kb deletion in the 4kb HindIII/EcoRI fragment
1
You draws restriction maps for the normal patient
6 8 2E H E4
You draws restriction maps for the affected individual
41
Complex Mapping
Often maps are more complex and difficult to analyze using single and double digests alone.To simplify the analyses, you can isolate each EcoRI band from the gel and then digest with HindIII
1
2
4
6
14
Mark
er
EcoR
IH
ind
III
12
EcoR
I+H
ind
III
1
2
4
6
14
Mark
er
12
1
2
4
6
14
12
1
2
4
6
14
12
Mark
er
Mark
er
12
kb
12
kb
+H
ind
III
6kb
6kb
+H
ind
III
2kb
2kb
+H
ind
III
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Mt DNA
Y chromosomes can be used to study paternal lineagemtDNA can be used to follow maternal lineage
Cells contain organelles- Mitochondria are organelles that produceEnergy. They contain a small 17,000 bp circular DNA.It encodes for 13 proteins in human cells and some tRNA’s
Hypervariable region (150 bp)
tRNA
NADH dehydrogenase
cytochromeC oxidase ATP synthase
CytochromeB
Mitochondrial DNA inheritance is not mendelianIt is inherited maternally
43
Using DNA to study History
This hypothesis was initially derived from restriction maps of mitochondrial DNA
All humans are derived from a small African population about 170K yrs ago
“Eve’s DNA”
Africa
Australia
Europe
Asia
44
Eve
Geographic region
DNA
Mutation generates B from A. Now you have individualsWith A and B DNA in population.
A
AB AB
C
D
45
Out of Africa
AB
C
D
C migrates to form a separate population.Additional mutations diversify DNAs in populations.Original population more diverse than newer population
AB
C
D
EC
F
G
Compared sequences of mtDNA
There are greater sequence differences among Africans thanany other group (Europeans, American Indians, Asians, etc etc)The african population had the longest time to evolve variationAnd thus humans originated in Africa.
48
Cloning DNA
A reasonable question is how did we clone a fragment of DNA
Or how do we clone a gene
The construction of Recombinant DNA molecules or cloning of DNA molecules
Recombinant DNA is generated through cutting and pasting of DNA to produce novel sequence arrangements
Restriction enzymes such as EcoRI produce staggered cuts leaving short single-stranded tails at the ends of the fragment. These “cohesive or sticky” ends allow joining of different DNA fragments
When a piece of DNA is cut with EcoRI, you get
|nnnGAATTCnnnnnnCTTAAGnnn |
nnnG AATTCnnnnnnCTTAA Gnnn
49
Cloning DNA
A reasonable question is how did we get the 20kb fragment of DNA in the first place?
To understand the origin of the fragment we must address the issue of:
The construction of Recombinant DNA molecules or cloning of DNA molecules
Recombinant DNA is generated through cutting and pasting of DNA to produce novel sequence arrangements
50
Recombinant DNA
Restriction enzymes such as EcoRI produce staggered cuts leaving short single-stranded tails at the ends of the fragment. These “cohesive or sticky” ends allow joining of different DNA fragments
When a piece of DNA is cut with EcoRI, you get
|GAATTCCTTAAG |
AATTC-----------------G G-----------------CTTAA
AATTC-----------------G G-----------------CTTAA
5’AAAAAAAAAAGGGGTTTTTTTG AATTCAAAAAAAAAAAAAAGGGGGGGGTTTTTTTG AATTCAAAAAAAAGGGGGGGGTTTTTT3’3’TTTTTTTTTACCCCAAAAAAACTTAA GTTTTTTTTTTTTTTCCCCCCCCAAAAAAACTTAA GTTTTTTTTCCCCCCCCAAAAAA5’
51
Plasmids
Plasmids are naturally occurring circular pieces of DNA in E. coli
The plasmid DNA is circular and usually has one EcoRI site. It is cut with EcoRI to give a linear plasmid DNA molecule
AATT
AATT
52
Plasmids
Small circular autonomously replicating extrachromosomal DNA
Bacterial genome(5000kb)
Plasmid DNA(3kb)
Modified plasmids, called cloning vectorsare used by molecular biologists to isolatelarge quantities of a given DNA fragment
Plasmids used for cloning share three properties
Unique restriction site
Antibiotic resistance
Origin of replication
E B
Origin
Antibiotic resistance
gene
53
Plasmid elements
Origin of replication:This is a DNA element that allows the plasmid to be replicated and duplicated in bacteria. Each time the bacterium divides, the plasmid also needs to divide and go with the daughter cells. If a plasmid cannot replicate in bacteria, then it will be lost.
54
Plasmid elements
Antibiotic resistance:This allows for the presence of the plasmid to be selectively maintained in a given strain of bacteria
Lab bacterial strains are sensitive to antibiotics.
When grown on plates with antibiotics, they die.
The presence of a plasmid with the antibiotics resistance gene allows these lab strains to grow on plates with the antibiotic. You are therefore selecting for bacterial colonies with the Plasmid
-antibiotics+antibiotics
55
Plasmid elements
Unique restriction sites:For cloning the plasmid needs too be linearized. Most cloning vectors have unique restriction sites. If the plasmid contains more than one site for a given restriction enzyme, this results in fragmentation of the plasmid
Why does this matter?
Antibiotic resistance gene
Ori
56
pUC18
pUC18 is one of the most commonly used plasmid:pUC= plasmid University of California
Plasmid replicon copy NopBR322 pMB1 15pUC18 pMB1 500pACYC p15A 10pSC101 pSC101 5
57
Cloning DNA
When a piece of DNA is cut with EcoRI, you get
|GAATTCCTTAAG |
AATTC-----------------G G-----------------CTTAA
AATTC-----------------G G-----------------CTTAA
-----------------G-----------------CTTAA
AATTC----------------- G-----------------
When tow pieces of DNA cut with EcoRI are ligated back together you get back an EcoRI site
59
Ligation
AATT
AATT
PLASMID
GENOMIC DNA
The EcoRI linearized PLASMID DNA is mixed with HUMAN DNA digested with EcoRI
The sticky ends will hybridize/anneal specifically and a recombinant plasmid will be generated
AATT
AATT
TTAA
TTAA
TTAA
TTAA
TTAA
TTAA
TTAA
TTAA
TTAA
TTAA
TTAA
TTAA
60
Cloning
d E F G h
The genomic DNA fragments is mixed with a plasmid that has been linearized at a single EcoRI site (say pUC18)
Ori
Am
pr
Both the plasmid and genomic DNA have been cut with EcoRI, they have complementary sticky ends
|G A A T T CC T T A A G |
AATT----------------------------- -----------------------------TTAA
Genomic DNA
________________________________________________TTAA
Plasmid
AATT
61
Recombinant plasmid
This process where foreign genomic DNA is joined to plasmid DNA is called ligationIt results in recombinant plasmid (foreign DNA+plasmid)Each plasmid has one foreign EcoRI fragmentEach foreign fragment is still present as only one copy! This is not useful.
Incompatibility of sticky ends
62
|G A A T T CC T T A A G |
AGCT--------------------------- ---------------------------TCGA
Genomic DNA
__________________________________________TTAA
Plasmid
AATT
Plasmid cut with EcoRI
Genomic DNA cut with HinDIII |A A G C T TT T C G A A |
____________________________________TTAA AATTAGCT-----------------------------
-----------------------------TCGA
Won’t work!!
63
TransformationThe entire collection of these plasmids bearing genomic DNA inserts is called a Genomic Library!These plasmids are added back into bacteria by a process called transformation
The bacteria are selected for the presence of the Plasmid by growth on media containing antibiotics
OriA
mp
r
Gene
Each colony of E. coli will harbor one plasmid with one piece of genomic DNA. Only cells with plasmid will grow on plates with antibiotics (the antibiotic resistance gene on plasmid allows these cells to grow). Cells that did not take up a plasmid will not grow.
d E F G h
Petri dish + antibiotic
64
Plasmid propagation
The plasmid DNA can replicate in bacteria and therefore many copies of the plasmid will be made. The human DNA fragment in the plasmid will also multiply along with the plasmid DNA.
THE DNA IS CLONED
Normally a gene is present as 2 copies in a cell. If the gene is 3000bp long there are 6x103 bp in a total of 6x109 bp of the human genome
Once ligated into a plasmid, unlimited copies of a single gene can be produced.The process of amplifying and isolating the human DNA fragment is called DNA cloning.
65
Why are plasmids important?
Most genes are present as two copies in the entire genome.Plasmids allow us to obtain 1000’s of copies of a gene in a pureform
Cloning and Expression of genes in cells
Coding region of protein
Enhancer, Promoter, Ribosome binding site
E B
Cloning genes
No cloning of RNA into double stranded plasmid DNA
No cloning of single stranded DNA into double stranded plasmid DNA
69
Isolate the plasmid
To isolate the gene fragment, we grow up a large population of E. coli containing the plasmid with the gene insert.
A simple procedure allows us to isolate the plasmid (which is smaller than Chromosomal DNA)
Once we have purified the plasmid we have 1000’s of copies of Gene in a plasmid
We can take the plasmid and cut it with EcoRI. When the digest is run on an agarose gel, we get two bands- one corresponding to the plasmid and one to the insert.
The DNA present in the band corresponding to the insert can be isolated from the gelPURE GENE !!!!!
Mark
er
EcoR
I
Un
cu
t
plasmid
Gene C
Foreign gene expression
70
Influenza virus promoter sequences do not work in chicken cells
Connect Influenza antigen gene to a chicken enhancer/promoter
ChickenPromoter
Influenza GeneChicken Enhancer
What if you want to express Influenza antigen in chicken cells?
Mixing and matching
71
Coding region GLOBIN gene
5’UTR
3’UTR
HinD
HinD
Blood specific promoter
Liver specific promoter
HinD
Kanr
ori
Globin Expression in liver
HinD HinD
Kanr
ori
72
Inter-species Gene transfer
Human Cell is cf-/cf-It becomes CF+ after transfection
CF gene on a plasmid
Isolate Plasmid
Transfect human cell with CF+ plasmid
CF+
Definition of Key Terms
73
CisgenicsGenetic modification of a recipient plant/animal with a gene from a sexually compatible plant/animal species
TransgenicsGenetic modification of a recipient plant with a gene from a sexually incompatible plant/animal or other organism
Traditional breedingConventional cross breeding of two species of plants to transfer a gene from one species to the other
Are cisgenics acceptable?
Are transgenics acceptable?
75
Genetically modified organisms GMO
Attempts to cross wheat and rye produce sterile offspring.
New techniques were developed that allowed production of fertile hybrid.
The two plants were treated with a potent toxin colchicine and the genomes were severely MUTAGENIZED
The mutagenesis allowed the genome of wheat and rye (these are different species) to overcome the species barriers, fuse and form a NEW SPECIES !!
These plants were used to develop genetically novel plants with traits from wheat and rye parents producing a “SuperFood”
GOOD IDEA?
BAD IDEA?
76
Inactivating single genes
Ethylene gas released by fruit accelerates the ripening process. Prevention of ethylene production would block the fruit from ripening prematurely and spoiling on the way to the market.
The ethylene biosynthetic pathway is as follows:
Precursor----->ACC------>ethylene
ACC ACC
synthase oxidase
Technology was used to generate mutants in the plant so that they could not synthesize the enzymes required for ethylene gas production.
Generated and marketed the “SUPER SAVR TOMATO”
GOOD IDEA?
BAD IDEA?
77
Expressing a foreign gene
A species of bacteria produces a potent natural pesticide
This pesticide is used in organic farming
The gene necessary for producing the toxin was identified and cloned.
The gene was inserted into the genome of plants.
This bacterial gene was now able to replicate in plants and the plant made and secreted the toxin. The plant now produced the toxin thus eliminating the need for pesticide spraying.
This reduces the harmful effects of pesticides on humans
However, insects start becoming resistant to this toxin.
GOOD IDEA?
BAD IDEA?
78
Reintroducing WT gene
Ancient native corn (roots) emit a volatile substance, b-caryophyllene, when attacked by insects. The substance attracts nematodes to the roots. These worms eat the insects protecting the corn.
Commercial corn has a mutation and cannot produce b-caryophyllene.
The wild type gene b-caryophyllene synthase was cloned.
A commercial corn plant was transformed with the wild type gene -b-caryophyllene synthase. The plant could now produce b-caryophyllene and was resistant to insects.
Good Idea
Bad Idea?
Ancient corn x commercial corn
Small cob large cobLarge height short heightInsect resistant insect sensitiveSlow growth rapid growthEasily stressed stress resistant
79
Is the trait present in close relatives
YesConventional breeding or CisgenicsParent1 x Parent2(start with ~1000 crosses)
F1 Phenotype selection(500,000 plants)
F2(50,000 plants)
F3(asses using markers)
F4(5000 plants)
F5(1000 plants- check yields, other traits)
F6(5 plants- submit for official trials)
(Linked genes along with trait desired)
or CisgenicsInsert single gene lost from ancestor
NOTransgenics(insert gene from other organisms)
80
Genetically modified plants
Trititcale- created in the 1880’s-1930’s by the Edinburgh Botanical Society. Using chemical mutagenesis combined with Mendelian crosses. It is currently sold primarily in organic health food stores. Is it a good idea to mutate crops using Is it a good idea to mutate crops using chemical mutagens?chemical mutagens?
Flavor Savr tomato helps transport fragile food preventing waste. Labeled a Frankenfood. It has a single mutation in one gene. Is it a good idea to mutate crops using recombinant DNA Is it a good idea to mutate crops using recombinant DNA methods? methods? What if you made the same mutation by classical genetics?
Bt cotton created in the 1990’s using recombinant DNA and transgenic technology. What if you inserted a gene from one species in to another species using classical genetics?
Reinserting Caryophyllene synthase into corn restores its natural insect resistance which was lost when commercial corn varieties were generated by classic breeding techniques. What if you inserted this gene back by genetic crosses?
Gene blocking may produce tea, coffee without the caffeineTomatoes with a higher antioxidant (lycopene) contentTomatoes with a higher antioxidant (lycopene) contentFungal resistant bananas Fungal resistant bananas Smaller, seedless melons for use as single servingsSmaller, seedless melons for use as single servingsBananas and pineapples with delayed ripening qualitiesBananas and pineapples with delayed ripening qualities
http://www.nytimes.com/2013/03/19/science/earth/research-to-bring-back-extinct-frog-points-to-new-path-and-quandaries.html?pagewanted=all
Message: Understand the differences (GM-foods) pre- and post-1990
Genomic clones
Genomic DNA
Digest with restriction enzyme
Ligate with plasmid
Grow individual plasmids in E. coli
Genomic DNA
Digest Genomic DNA and plasmid with restriction enzyme
Ligate with cut plasmid DNA
Grow individual plasmids in E. coli
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Gene1 Gene2 Gene3
A B C D E F
Each fragment is ligated into the plasmid
Each plasmid is put (transformed) into E.coli
Each E. coli colony on a plate has one specific plasmid
A
B
C
D
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Genomic clone libraries
Species Genome size average #plasmids insert size
E. Coli 5000kb 16 kb 1300Drosophila 150,000kb 16 kb 46,000Human 3000,000kb 16 kb >100,000
An entire genome of any organism can be cloned as small fragments in plasmids
The larger the genome, the more difficult the task
At present, genomic DNA libraries exist for a large number of organisms including
Yeast, C.elegans, Drosophila, Zebrafish, Xenopus, Chickens, Mouse, Humans etc
RNA Cannot be cloned
Reverse transcriptase copies RNA into DNA
So to clone RNA, you first convert RNA into DNA using reverse transcriptase
This DNA (cDNA) is an complementary copy of the RNA (RNA was the template)
The cDNA is then cloned into plasmids
cDNA clone
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cDNA
Often we have RNA rather than DNA as the starting materialFor instance in the case of the human hemoglobin gene, we started with globin mRNA
RNA is difficult to work with. In contrast to DNA, RNA breaks down and degrades very easily. There are no restriction enzymes that cut RNA at specific sites.RNA cannot be cloned. It cannot be inserted into a plasmid and amplified since all Plasmids are DNA.
The enzyme reverse transcriptase has proven very useful to molecular biologists.This enzyme catalyzes the synthesis of DNA from a RNA template. It is normally found in a large class of viruses. The genome of these viruses is RNA!! These viruses are called retroviruses.They infect eukaryotic cells and use these cells to grow/replicate
Retroviruses carry an RNA genome. Interestingly they will integrate into the DNA of the host. For RNA to integrate into DNA, first the RNA has to be converted to DNA
Remember the central dogma of molecular biologyInformation flows from DNA to RNA to protein!
DNA---->RNA---->protein
Reverse Transcriptase reverses this dogma (partially)
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cDNA/splicing
So from globin mRNA, a complementary DNA molecule can be created using reverse Transcriptase. This complementary DNA is called cDNA.The cDNA can now be inserted into a plasmid and cloned.
What is the relationship between a cDNA clone and a genomic clone?
SplicingIn eukaryotes, the coding sequences are interrupted by introns
Gene7700 nt
1 2 3 4 5 6 7
Ovalbumin
Primary transcript
Splicing
mRNA1872 nt
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Genomic clones represent the organization of the DNA in the nucleus!
cDNA clones represents the organization of mRNA sequences after the gene has been transcribed, processed and exported to the cytoplasm.
cDNA clones contain the sequence of nucleotides that code for the mRNA--protein!
cDNA clones do not contain the sequence of the promoter of the gene or the intron.
The starting material for cDNA clones is different from material used to make genomic clones
Genomic clone cDNA clone
Source Nucleii cytoplasmic RNA(any cell) (specific
cell type)
Use Studies on gene Studies directed
organization & towards coding regions
structure
Genomic Vs cDNA
PCR
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It’s a method that can be used to make many copies of a particular DNA sequence from a particular individual
You have to know the DNA sequence before you can amplify that sequence (it does not have to be cloned)
The sequence will not propagate (replicate) in living organisms
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PCR
Heat 95C to denature DNA and add primers
Let Primers hybridize to DNA (55C)Add Heat resistant DNA polymerase and dNTP (70C)
Repeat- 95C55C70C
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5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
PRIMER1 5’AAAGATC3’
3’AGCTAGAT5’ PRIMER2
5’AAAGATC3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’AGCTAGAT5’5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
3’TTTCTAGCCCCCCCCCCCCCCC
GGGGGGGGGGGGGGGTCGATCTA3’5’AAAGATC
AGCTAGAT5’
How do you detect PCR?
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Size of PCR product will depend upon location of PCR primers
Agarose Gels
PC
R
PCR cloning
IF YOU KNOW THE SEQUENCE OF THE GENE YOU WANT TO CLONE
You can use PCR to first make many copies of your geneThen you cut the PCR fragment and plasmid with a restriction enzymeLigate PCR with plasmid, transform E.coli
Then you can clone those copies into a plasmid.
PCR clone
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You can Restriction map a cloned piece of DNA
Can you restriction map a piece of DNA in the genome without first cloning it?
Blotting
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Southern blotting
Rapid method of identifying a specific DNA fragment from amixture of fragments (or from different individuals)
Mark
er
EcoR
I
Un
cu
t
How do you determine which band corresponds to insert and which to the plasmid
EcoRI
plasmid
InsertGene
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A probe is used to identify genomic DNA?
DNA is transferred from the gel to a paper filter
The DNA (plasmid and chromosomal) on the paper is denatured (converted from ds to ss)
Then you take the filter and to it add radiolabeled probe (small part of Gene).
Mark
er
EcoR
I
Un
cu
t
Mark
er
EcoR
I
Un
cu
t
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Southern blotting with a probe
The probe AAAAAAA will bind the single stranded DNA that has a complementary sequence (TTTTTT). It will specifically hybridize with the insert (genomic DNA)
A probe with this specific sequence is generated and made radioactive
Incubate the filter with the radio-labeled probe
A specific probe enabled us to identify a DNA fragment that corresponds to a specific gene of interest.
Mark
er
EcoR
I
Un
cu
t
gatcgatcgatcTTTTTTTgatcgatc AAAAAAA
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PROBES
Probes are obtained in a number of ways
RNA as a sourceThe probe for hemoglobin can be obtained from mRNA of immature red blood cells.The major transcript of these cells is from the hemoglobin gene. So isolating RNA from these cells, we can obtain a relatively pure probe for the hemoglobin gene
ProteinIf you have a purified protein, the amino acid sequence can be determined.From the amino acid sequence, using the genetic code a corresponding DNA sequence can be synthesized and this small DNA piece can be used as a probe
HomologyProbes from conserved genes-Many genes are conserved from one species to anotherChimpanzee and human DNA are 97% identical. If you know the sequence of a gene in chimps, then you will be able to know the sequence for the gene in humans!The histone genes are highly conserved across phyla. Histone proteins have three Amino acid differences between humans and peasHistone genes have been isolated in yeast, they can serve as probes for screening a Human genomic library- cloning by phone
The computer databases
PCR
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What about a genome?
What if Gene C was in a large genome. Could we identify the fragment by Southern blotting
Mark
er
Mark
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Transfer tomembrane
Mark
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Hybridize withProbe C
EcoRI
1kb 2kb 3kb 4kb 5kb 4.5kb 0.5kb
GeneC GeneX GeneAGeneR
Based on the blot what is the restriction map for gene C?
Map by Blotting
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Southern Blot inferred Map
Actual Map
4kb
GeneC
E E
1kb 2kb 3kb 4kb 5kb 4.5kb 0.5kb
GeneC GeneX GeneAGeneR
H H
E E E E E E
3kb 8kb 9kb
Mark
er
Mark
er
EcoRI
You can build a more detailed genomic restriction map
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1kb 2kb 3kb 4kb 5kb 4.5kb 0.5kb
GeneC GeneX GeneAGeneR
H H
E E E E E E
3kb 8kb 9kb
Southern Blot inferred Map
4kb
GeneC
E E
H H8kb
Mark
er
HindIII
Mark
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EcoRI
Mark
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Mark
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You can build a more detailed genomic restriction map
If we digest the DNA with HindIII and EcoRI what will happen?
Southern Blot inferred Map
4kb
GeneC
E E
H H8kb
Mark
er
HindIIIEcoRI
Mark
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EcoRI+ HindIII
Mark
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GeneC
ProbeAProbeB
Mapping chromosomal DNA with different probes
Probe A 2Kb fragment
ProbeC
E E E E
1kb 2kb 4kb 3kb
E
Probe B 2Kb and 4Kb fragment
ProbeC 4Kb fragment
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Restriction mapping Individuals
Mark
er
Mark
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Gene
E E E E1 2 4
A B
Mark
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Mapping deletion with probe B.
Gene
E E E E1 1 4
AB
WT Mutant WT
Mark
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Mutant
Mapping deletion with probe A.
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Northern blot
This is a rapid method that allows you to determine the cell type in which a specific gene is active and being transcribed.
Presence of RNA is a reflection of gene activity
Embryo
Brain
Bone
Blood
Lung
liver
These tissues differ because each is transcribing a unique subset of genes.
Each tissue contains a unique and distinct mRNA population
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Northern blot
Method is analogous to Southern blotsInstead of DNA as the starting material, you use RNA.You take cells, break them open, isolate the RNA and run the RNA on a gel
Transfer RNA to membrane and use probe for gene of interest.
The RNA can be from specific tissues or cell types
Presence of RNA is a reflection of gene activity
Lym
ph
ocyte
Bra
in
ery
thro
cyte
Bon
e m
arr
ow
Kid
ney
WT individualGlobin probe
Lym
ph
ocyte
Bra
in
ery
thro
cyte
Bon
e m
arr
ow
Kid
ney
mutant individualGlobin probe
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Microarray Microarrays are miniature devices containing thousands of DNA sequences stuck on at different positions (addresses). Hybridisation to complex mixtures of labelled DNA molecules, prepared from cellular RNA, shows the relative expression levels of thousands of genes. This can be used to compare gene expression levels within a sample or look at differences in the expression of specific genes across different samples.Key principles
* RNA isolated from a particular cell type or tissue comprises a complex mixture of different RNA transcripts. The abundances of individual transcripts in the mixture reflect the expression levels of the corresponding genes.
* A microarray is a small device, about the size of a microscope slide, with thousands of different known DNA sequences immobilised at different addresses on the surface.
* Each of these DNA sequences can participate in a hybridisation reaction.
* If a complex DNA mixture copied from the above RNA is labelled and hybridised to the microarray, the strength of the signal at each address shows the relative expression levels of the corresponding gene.
Microarrays can be used to compare gene expression levels within a sample or look at differences in the expression of specific genes across different samples.How does it work?
Single DNA strands with complementary sequences can pair with each other and form double-stranded molecules. This hybridisation process occurs with such specificity that a labelled DNA probe - a single DNA strand carrying a label that allows it to be detected — can pick out a matching partner, the target, in a complex mixture containing millions of different sequences.
Microarrays apply the hybridisation principle in a highly parallel format. Instead of one target, thousands of different potential targets are arrayed on a miniature solid support. Instead of a unique labelled DNA probe, a complex mixture of labelled DNA molecules is used, prepared from the RNA of a particular cell type or tissue.
The abundances of individual labelled DNA molecules in this complex probe reflect the expression levels of the corresponding genes. When hybridised to the array, abundant sequences will generate strong signals and rare sequences will generate weak signals.
The strength of the signal thus represents the level of gene expression in the original sample.How is it used?
Expression analysis with microarrays can be used to determine what genes are expressed in a particular cell type or tissue and to compare the expression levels of different genes.
It can also be used to compare gene expression across different but related samples, such as disease vs healthy tissue. A gene expressed only in the disease sample, for example, might represent a useful drug target. Comparative expression analysis can be achieved by comparing duplicate microarrays hybridised to complex probes prepared from the alternative samples.
A refinement of the technique, in which RNA from the related samples is labelled with different fluorescent molecules, allows this analysis to be carried out on a single microarray.
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MicroarraysThese are reverse northern blots.
Allows us to examine gene expression of all of the genes in the genome!
Each spot is DNA for one defined gene.
Each gene DNA is spotted in a grid.
They cover the entire genome.
Make total RNA from normal and mutant cell,Label each total RNA differentlyWt=redMut=greenAdd labeled RNA from normal and mutant cells to array and let hybridizeMeasure label and determine change
Ratio of WT/mut
WTMut
1
2
3
4
51, 2, 3 … are sequences specific for gene1, gene2, gene3 etc printed on the slide
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Use of microarrays
To measure changes in transcription of genes during drug treatmentTo identify deletions in DNA
A microarray works by exploiting the ability of a given mRNA molecule to bind specifically to, or hybridize to, the DNA template from which it originated.
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Genome sequencing
Whether bacterium or human, the genome of any organism to too large to be deciphered in one go. The genome is therefore broken into smaller pieces of DNA, each piece is sequenced and computers fit all the sequences back together.
The human chromosome to be sequenced.The chromosome is first chopped randomly into conveniently sized chunks.These large fragments are inserted into bacterial artificial chromosomes (BACs) and cloned in bacteria.These fragments are then mapped so it is known which region of the chromosome they came from.Each BAC is shotgunned - broken randomly into many small pieces. This process is repeated several times to give different sets of fragments. (The whole-genome shotgun method goes directly to this stage.)The fragments are cloned in small vectors and then sequenced. About 500 bases of sequence information is produced from each fragment.The sequences are fed into a computer, which looks for overlaps at the end of the sequence to find neighbouring fragments.When many fragments have been sequenced the sequence of the original BAC insert can be assembled. The process is carried out for all the BACs to give a complete chromosomal sequence.For example, the human genome is about 3 billion base pairs, arrayed in 24 chromosomes. The chromosomes themselves are 50–250 million bases (megabases) long. These tracts of DNA are much too large for even the latest automated machines, which sequence fragments of DNA between 400 and 700 bases long.The genome is first broken into conveniently sized chunks, fragments of about 150 kilobases. Each fragment is inserted into a bacterial artificial chromosome (BAC), a cloning vector used to propagate DNA in bacteria grown in culture.The BACs are then mapped, so that it is known exactly where the inserts have come from. This process makes re-assembling the sequenced fragments to reflect their original position in the genome easier and more accurate, and any one piece of human DNA sequence can automatically be placed to an accuracy of 1 part in 30 000.Each of the large clones is then 'shotgunned' - broken into pieces of perhaps 1500 base pairs, either by enzymes or by physical shearing - and the fragments are sequenced separately. Shotgunning the original large clone randomly several times ensures that some of the fragments will overlap; computers then analyse the sequences of these small fragments, looking for end sequences that overlap - indicating neighbouring fragments - and assembling the original sequence of the clone.An alternative approach, 'whole genome shotgun sequencing', was first used in 1982 by the inventor of shotgun sequencing, Fred Sanger, while working on phages (viruses of bacteria). As its name suggests, in this technique the whole genome is broken into small fragments that can be sequenced and reassembled. This method is very useful for organisms with smaller genomes, or when a related genome is already known.
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Animal cloning
Animal clones are genetically identical. Natural clones occur in the form of identical twins but it is also possible to produce artificial clones by nuclear transfer. The nucleus is removed from a somatic (body) cell and placed in an egg whose own nucleus has been removed. The egg is then implanted in a surrogate mother and develops to term.
Key principles * Differentiated animal cells are unable to develop into complete animals *The nuclei of most differentiated cells retain all the necessary genetic information. * Transfer such a nucleus into an egg whose own nucleus has been removed. * Transfer to the environment of the egg reprograms the nucleus (makes it forget its history) and allows the full development of a viable animal that is genetically identical to the donor of the somatic cell. * Until 1997, cloning in mammals was only possible using nuclei obtained from very early embryos. A breakthrough was made when cloning was achieved using nuclei from adult cells. * Recent research suggests that animals produced by cloning from adult cells may age prematurely, but further investigation is necessary.
How does it work?Nuclear transfer is carried out by fusing the donor somatic cell to an egg whose own nucleus has been removed. Fusion is achieved in a culture dish by applying an electric current. The change in electrical potential also mimics the normal events of fertilisation and initiates development.A key aspect in the success of nuclear transfer is synchronisation of the cell cycles between the donor nucleus and the egg. Before fertilisation, the egg's nucleus is quite inactive. The nucleus of the donor cell must also be made inactive otherwise it will not be reprogrammed and development will fail. Inactivation is achieved by culturing the cell but starving it of essential nutrients. The cell stops dividing and enters a quiescent state compatible with nuclear transfer.How is it used?Animal cloning has the potential to overcome the limitations of the normal breeding cycle. In the future, it may be used to produce elite herds by cloning the superior animals, or to rapidly produce herds of transgenic or otherwise modified animals. Transgenic farm animals make useful bioreactors, producing valuable proteins in their milk.Another application is the use of genetically-modified pigs as a source of organs suitable for transfer to humans (xenotransplantation).
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How is a specific gene isolated (CLONED)?
Its like going to the library and looking for a specific book.
It involves screening through a genomic library.A genomic library is a large collection of plasmids containing pieces of DNA from a specific species. The set of cloned fragments is so comprehensive that virtually the entire genome is represented in the library.
The fragments that make up the library are initially generated by digesting genomic DNA (e.g. human) with a restriction enzyme- say EcoRI
The EcoRI sites are randomly distributed in the genome- fragments of varying lengths will be generated.Some fragments will contain one gene, others two genes or cut genes in half.
Gene1 Gene2 Gene3
A B C D E F
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Gene1 Gene2 Gene3
A B C D E F
Each fragment is cloned into the plasmid, each plasmid is put(transformed) into E.coli
A
B
C
D
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The library is random!
Each fragment is cloned into the plasmid, each plasmid is put(transformed) into E.coli
Gene1 Gene2 Gene3
A B C D E F
A
B
C
D
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Fragments,bookmark, title
The library is not bookmarked or even titled and is in fragments!There is no organization to the library. It is simply a populations of cloned fragments representing the entire genome.
The equivalent of this would be if you went to the University Library to find all the books in a large heap, the books had no title, and in addition instead of entire books you often found parts of books.
How do you use such a library? How do you find the book you are interested in.
Lets work our way through this problem with a simple example
Organism has EIGHT genes in its genome
A B C D E F G H
EcoRI
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Genomic library
If we wanted to study gene C- Create a restriction map of gene CDetermine it sequenceStudy proteinC
What do we need to do
We need to initially clone the gene and make many copies of gene C
Creating a genomic library provides a means of obtaining many copies of gene C
To generate a genomic library:Total genomic DNA is isolated from the species of interestThe DNA is cut with EcoRI
A B C D E F G H
A b
b C d
d E F G h
h
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Genomic library
A b
b C d
d E F G h
h
These genomic DNA fragments are mixed with a plasmid that has been linearized at a single EcoRI site (say pUC18)
Ori
Am
pr
Ori
Am
pr
Ori
Am
pr
Ori
Am
pr
Both the plasmid and genomic DNA have been cut with EcoRI, they have complementary sticky ends
|G A A T T CC T T A A G |
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Recombinant plasmid
This process where foreign DNA is joined to plasmid DNA is called ligationIt results in recombinant plasmid (foreign DNA+plasmid)Each plasmid has one foreign EcoRI fragmentEach foreign fragment is still present as only one copy! This is not useful.
d E F G h
b C d
A b
h
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How are genomic libraries used?
If we are interested in studying gene C, you need the plasmid containing gene C
Having a genomic library means you have gene C, but where is it? Which colony on the Petri dish contains gene C?
Genomic libraries are much more complex than the one described for our hypothetical 8 gene organismYou need to identify one recombinant plasmid out of 100,000’s present in a library.
Identifying and isolating a specific plasmid is called screening a library.This requires a probe
A probe is a sequence complementary to PART of the sequence one wishes to pull out.
You radiolabel the probe and once labeled the probe is used to identify the plasmid containing E. coli colony
How do we get the probe?
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The genomic library and a specific probe enabled us to achieve two goals
Out of the billions of base pairs in a large genome, we have been able to identify a few 1000 base pairs that correspond to a specific gene of interest.
In addition we were able to isolate this sequence on a specifically engineered plasmidThat allows us to make large quantities of this rare sequence.
Genomic libraries are described in terms of average fragment size and the number of plasmids that must be screened to have the entire genome represented
To have a good probability (>99%) of identifying a given DNA sequence (gene) present in the collection of plasmids (library). The number of plasmids (colonies) that must be screened is a function of the size of the genome of the species from which the Library was constructed.