pharm biotech cloning and mutation class

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DNA Cloning and MutationDNA Cloning

and MutationBT-510 Biotechnology in Pharmaceutical

Sciences

Dr Sanjay K BanerjeeDr Sanjay K Banerjee

Pharmacology DivisionPharmacology Division

Indian Institute of Chemical Technology (IICT)Indian Institute of Chemical Technology (IICT)



Joining DNA molecules.

mopolymer tailing.

lunt end ligation.

Sticky end ligation.e methods exist for joining DNA molecules in vitro.

- DNA fragments cloned and manipulated within“Vectors”

Human DNA

DigestIsolate

fragment

Jointogether

Vector

DNA

Recombinantconstruct

Often called aninsert

Vectors include plasmids or viruses which allow

sequence amplification.



In molecular biology, a vector is a DNA molecule used as a vehicle totransfer

foreign genetic material into another cell.

The four major types of vectors are 1. plasmids, 2. bacteriophages and other viruses, 3. cosmids, and

4.artificialchromosomes.All engineered vectors have an origin of replication, a multicloning site,

and a selectable marker.

Vector

1. Antibiotic resistant screening

2. Blue/white screeningX-gal is cleaved by β-galactosidase yieldinggalactose and 5-bromo-4-chloro-3-hydroxyindole. The latter is then oxidizedinto 5,5'-dibromo-4,4'-dichloro-indigo, aninsoluble blue product. Thus, if X-gal and aninducer of β-galactosidase (usually IPTG)is contained within an agar medium on aculture plate, colonies which have a functio

lacZ gene can easily be distinguished.



-Both E.coli and T4 phage encode DNA ligaseenzyme that seals single stranded nicks

between adjacent nucleotides.

1.Sticky end ligation;

Vector DNAForeign (Insert) DNA

“Sticky end”ComplementaryBase pairing



-“Sticky end” ligation is easiest way of joiningDNA.

(ii) Treating vector with an Alkaline Phosphatase(Alk Phos.)

(i) Manipulating concs. and ratios of the vector /insert DNA to favour formation of recombinantmolecules.

-Reaction manipulated to favour production of recombinant molecules in following ways;

-Sticky end ligations done at 4-160

C to favourstability of hydrogen bonding.

-Removes the 5’ terminal phosphate sopreventing vector religation.



AlkalinePhosphatase2Pi

e Use of Alkalineosphatase for

eventing vectorligation

LigaseNo

Reaction

P

OH

P

OH

Vector DNA

Insert DNA

OH

OH P

P Ligase

OH

OH

OHOH

Transformation

(bugs grow)

Nick

Nick



2. Blunt end ligation.

-Involves adding long tails of adenine to onemolecule and a long tail of thymidines to anothermolecule using terminal

-Seldom used nowadays although sometimesused for cDNA cloning.

3. Homopolymer tailing.

-Often used to link “adaptors” containing stickyend restriction sites to blunt end of vector ortarget sequence.

s a practical limitation of 3-4 kb.

equires higher conc. of DNA.

erformed by T4 ligase.

uch less efficient.



PCR cloning-Two types;

-RE requires the presence of 4-8 base pairs fromend of molecule to cut DNA.

- Will work and introduce sites following first few

cycles of PCR.

-Non homologous 5’ ends added to primersdesigned for PCR.

Addition of sticky end restriction sites.

-Kits designed to allow rapid cloning of Taq PCRproducts

olymerase adds a single 3’ A overhang (Unique toT/A cloning.

-Use a vector with a single 3’T overhang.



Denature, anneal primers 1 and 2

C T T AAG C C G G

G C G C AAG C T T

PCR amplification with primers 1 and 2

Target Region

GCGCAAGCTT

CGCGTTCGAA CTTAAGCCGG

GAATTCGGCC

HindIII EcoRI



3. Always try your best to produce fragments withdifferent sticky ends (even one blunt end isbetter) as life becomes much easier.

2. Avoid same sticky end ligations.e.g. an insertwhich has two EcoRI ends.

1. Beware of cloning a blunt end insert biggerthan 3kb into a blunt end vector site.

Cloning tips.

When designing a cloning strategy it is worthknowing which types of ligation work the best.

4. When designing a cloning protocol several

small easy steps are easier than one big one!!!!!



Mutations are changes to the nucleotide

sequence of the genetic material of anorganism.

Mutations can be caused by copyingerrors in the genetic materialduring cell division,by exposure to ultraviolet,by ionizing radiation,by chemical mutagens,viruses,

also can be induced by the organism

Mutation



e-scale mutations in chromosomal structur

http://en.wikipedia.org/wiki/File:Types-of-mutation.png



Amplifications (or gene duplication) leading tomultiple copies of all chromosomal regions, increasingthe dosage of the genes located within them.Deletion of large chromosomal regions, leading toloss of the genes within those regions.

Mutations whose effect is to juxtapose previously

separate pieces of DNA, potentially bringing togetherseparate genes to form functionally distinct fusiongenes (e.g. bcr-abl). These include: Chromosomal translocations: interchange of

genetic parts from nonhomologous chromosomes.Interstitial deletions: an intra-chromosomal deletionthat removes a segment of DNA from a singlechromosome, thereby opposing previously distantgenes.



For example, cells isolated from a humanastrocytoma, a type of brain tumors, were foundto have a chromosomal deletion removing

sequences between the "fused in glioblastoma"(fig) gene and the receptor tyrosine kinase "ros",producing a fusion protein (FIG-ROS). Theabnormal FIG-ROS fusion protein has

constitutively active kinase activity that causesoncogenic transformation (a transformation fromnormal cells to cancer cells).

Chromosomal inversions: reversing theorientation of a chromosomal segment.Loss of heterozygosity: loss of one allele,either by a deletion or recombination event, in anorganism that previously had two different alleles.



all-scale mutations

l-scale mutations, such as those affecting a small gene in one or a fewotides, including:

int mutations, often caused by chemicals or malfunction of DNA replication,hange a single nucleotide for another.ilent mutations, which code for the same amino acid

issense mutations, which code for a different amino acid.onsense mutations, which code for a stop and can truncate the protein.

sertions add one or more extra nucleotides into the DNA. They are usuallysed by transposable elements, or errors during replication of repeating eleme

g. AT repeats).

letions remove one or more nucleotides from the DNA. Like insertions, thesetations can alter the reading frame of the gene.



effect on function:f-function mutations

of-function mutationsnant negative mutations l mutations k mutation or reversion

impact on protein sequence:

eshift mutationense mutationsral mutation

sense mutationt mutations

eficial mutationsions may have a positive effect given certain selective pressuresopulation.ample, a specific 32 base pair deletion in human CCR5 (CCR5-Δ32)

rs HIV resistance to homozygotes and delays AIDS onset in

ozygotes.



Somatic versus Germline mutation



utation and cancer

ancer is defined as the loss of cell division control.

ancer is caused by accumulation of many mutations withina single clone of cells.t least 5-6 genes must be mutatedrobably at least 100 genes which if mutated can cause cancer



Heredity and Cancer



Morphological mutants in

Drosophila



Dominant mutation

Recessive mutation

pharm biotech cloning and mutation class

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