1 basic techniques ---nucleic acid hybridization complementary strands will associate and form...

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’



GGCCCAAAAAAAAGGGGGGGGTTT3’ GTTTTTTTTCCCCCCCCAAA5’

SmaI AAACCCGGGAAA XmaI AAACCCGGGAAATTTGGGCCCTTT TTTGGGCCCTTT

EcoRI AAAGAATTCAAA MfeI AAACAATTGAAATTTCTTAAGTTT TTTGTTAACTTT

KpnI AAAGGTACCAAA Asp718 AAAGGTACCAAATTTCCATGGTTT TTTCCATGGTTT

Enzyme compatibility

xxxxxxx

18

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

28

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

31

Mapping

HindIII EcoRI HindIII/EcoRI14 12 86 6 6

42 2

32

Mapping


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

Another linear map

33

2

4

6

8

14

Mark

er

EcoR

I

Hin

dII

I

12

EcoR

I+

Hin

dII

I

1

PstI

EcoR

I+ P

stI

34

Different Mapping example

Hi Ec Hi/Ec12 12 88 6 6

42 2

Ps Ps/Ec13 127 5

21

Three different enzymesHiEcPs

35

Mapping


42 2

HindIII

12 & 8

12 & 8

36

Mapping


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

37

Mapping

EcoRI PstI PstI/EcoRI12 13 126 7 52 2

1

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

39

Final Map

2

5 1

4 8

P

E E H

Hi Ec Hi/Ec12 12 88 6 6

42 2

Ps Ps/Ec13 127 5

21

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

42

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.

Xxxxxx

46

47

THE GENE PROBE!!!

How do we isolate a GENE?

How did we get a pure copy of the gene?

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


|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


|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


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

E B

H

E E

BH

B

Coding region cloning

E E

H B

E E

H

S

K

Promoter cloning

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




xxxxxxxxx

81

What are genomic clones

What are cDNA clone

What is a PCR clone of a specific gene

Types of clones

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

85

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

86

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

88

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)

89

cDNA synthesis

Protein coat

RNA genome

Reversetranscriptase

mRNA

DNA

RT

DNA

mRNA

cDNA

90

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

91

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

92

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

93

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

95

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?

96

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

xxxxxxxxx

98

99

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

100

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

101

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

102

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

103

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

104

What about a genome?

What if Gene C was in a large genome. Could we identify the fragment by Southern blotting

Mark

er

Mark

er

Transfer tomembrane

Mark

er

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

105

Southern Blot inferred Map

Actual Map

4kb

GeneC

E E



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

106



H H

E E E E E E

3kb 8kb 9kb


4kb

GeneC

E E

H H8kb

Mark

er

HindIII

Mark

er

EcoRI

Mark

er

Mark

er

107

You can build a more detailed genomic restriction map

If we digest the DNA with HindIII and EcoRI what will happen?


4kb

GeneC

E E

H H8kb

Mark

er

HindIIIEcoRI

Mark

er

EcoRI+ HindIII

Mark

er

108

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

109

Restriction mapping Individuals

Mark

er

Mark

er

Gene

E E E E1 2 4

A B

Mark

er

Mapping deletion with probe B.

Gene

E E E E1 1 4

AB

WT Mutant WT

Mark

er

Mutant

Mapping deletion with probe A.

xxxxxx

110

111

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

112

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

113

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.

114

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

115

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.

116

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.

117

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).

xxxxxx

118

119

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

120

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

121

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

122

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

123

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

124

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


125

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

126

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?

127

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.

xxxxxx

128

1 basic techniques ---nucleic acid hybridization complementary strands will associate and form...

Documents