RECOMBINANT DNA

Presented by: Syeda Saba kazmi Dated:2/9/2015

OUTLINE

rDNA

• rDNA technology

• Basic techniques

• Restriction enzymes

• Vectors

• applications

Site directed mutagenesis

• About random mutagenesis

• Description of site directed mutagenesis

DNA sequencing

• Definition

• Sanger method

• Applications

• references

Genetic engineering & Biotechnology

• The modification of DNA of an organism to produce new genes with new characters

Genetic Engineering

• Use of living organisms to perform practical tasksBiotechnology

Recombinant DNARecombinant DNA

RE=again

Combinant=binding/combining

DEFINITION:Recombinant DNA (rDNA)

molecules are DNA molecules formed by bring together genetic material from multiple sources, creating sequences that would

not otherwise be found in biological organisms

Carry genetic information. Components of DNA are universal (Nucleotides), only the difference exists in arrangement of bases

rDNA is artificial new DNA that can be synthesized in vivo or vitro through biological techniques e.g. molecular cloning

DN

A

rDN

A

History of rDNA

Idea: Peter Lobban, Student of Biochemistry Department at Stanford University Medical School in 1972 -1973

Insulin (First drug for human), developed by Eli Lilly and Company 1980

Stanley N. Cohen and Herbert W. Boyer

Stanley N. Cohen (1935–) (top)

and Herbert Boyer (1936–)

(bottom), who constructed the

first recombinant DNA using

bacterial DNA and plasmids.

Stanley N. Cohen , who

received the Nobel Prize in

Medicine in 1986 for his

work on discoveries of

growth factors.

Creating recombinant DNA

Molecular cloning

• Molecular cloning is the laboratory process used to create recombinant DNA molecular cloning involves replication of the DNA within a living cell.

Polymerase chain reaction(PCR)

• PCR is the laboratory process used to create recombinant DNA that replicates DNA in the test tube, free of living cells.

Recombinant DNA Technology

DEFINITIONIntentional modification of organisms’ genomes for practical purposes

Three goals

Eliminate undesirable phenotypic traits

Combine beneficial traits of two or more organisms

Create organisms that synthesize products humans need

How is Recombinant DNA made?

There are three different methods by which Recombinant DNA is made.

TransformationPhage

IntroductionNon-Bacterial

Transformation

Transformation(Steps)

*select a piece of DNA to be inserted into a vector.

*cut that piece of DNA with a restriction enzyme

*ligate the DNA insert into the vector with DNA Ligase

*The vector is inserted into a host cell

One example of a possible host cell is E. Coli.

Phage Introduction

Phage introduction is the process equivalent to transformation, except a phage is used instead of bacteria.

E.g. lambda or MI3 phages to produce phage plaques which contain recombinants.

Non-Bacterial Transformation

very similar to Transformation

But does not use bacteria such as E. Coli for the host.

In microinjection, the DNA is injected directly into the nucleus of the cell being transformed.

or the host cells are bombarded with high velocity micro projectiles, such as particles of gold or tungsten that have been coated with DNA.

Applications of rDNA technology

Better Crops (drought & heat resistance)

Recombinant Vaccines (Hepatitis B)

Prevention and cure of sickle cell anemia

Prevention and cure of cystic fibrosis

Production of clotting factors

Production of insulin

Production of recombinant pharmaceuticals

Plants that produce their own insecticides

Germ line and somatic gene therapy

Insect-resistant tomato plantsThe plant on the left contains a gene that encodes a

bacterial protein that is toxic to certain insects that

feed on tomato plants. The plant on the right is a

wild-type plant. Only the plant on the left is able to

grow when exposed to the insects.

Transgenic animals

Green fluorescence Red fluorescence

Transgenic animals

A transgenic mouse

Mouse on right is normal; mouse on left is transgenic animal expressing

rat growth hormone

Cloned gene

Retrovirus

capsid

Bone

marrow

cell from

patient

Inject engineered

cells into patient.

Insert RNA version of normal allele

into retrovirus.

Viral RNA

Let retrovirus infect bone marrow cells

that have been removed from the

patient and cultured.

Viral DNA carrying the normal

allele inserts into chromosome.

Bone

marrow

Somatic cells

Only!

Not for

reproductive

cells !!

just a joke

Tools of Recombinant DNA Technology

• Bacterial enzymes that cut DNA molecules only at restriction sites

RESTRICTION ENZYMES

• Two groups based on type of cut

• Cuts with sticky ends

• Cuts with blunt ends

The Tools of Recombinant DNA Technology

• Nucleic acid molecules that deliver a gene into a cell

• Include viral genomes, transposons, and plasmids

Vectors

• Small enough to manipulate in a lab

• Survive inside cells

• Contain recognizable genetic marker

• Ensure genetic expression of geneare

4 different type of vectors:

Plasmid vectors

Lamda (λ) phage vectors

Cosmids

Expression vectors

The Tools of Recombinant DNA Technology

• A collection of bacterial or phage clones

• Each clone in library often contains one gene of an organism’s genome

Gene Libraries

• Library may contain all genes of a single chromosome

• Library may contain set of cDNA complementary to mRNA

Cont ….

Multiplying DNA in vitro:

• Large number of identical molecules of DNA produced in vitro

• Critical to amplify DNA in variety of situations in vivo

The Polymerase Chain Reaction

(PCR)

• Epidemiologists use to amplify genome of unknown pathogen

Cont ….

Multiplying DNA in vitro:

• Separates molecules based on electrical charge, size, and shape

• Allows scientists to isolate DNA of interest

Gel Electrophoresis

and the Southern Blot

• Negatively charged DNA drawn toward positive electrode

• Smaller fragments migrate faster than larger ones

Cont ….

What Is a Mutation?

Genetic information is encoded by thesequence of the nucleotide bases inDNA of the gene. The four nucleotidesare: adenine (A), thymine (T), guanine(G), and cytosine (C), a mutation is achange in the order of thesenucleotides.

A change in the order can cause thegene to encode for wrong proteins andinhibit the function of the gene or causethe gene to be virtually inactive.

Random mutagenesis

• based on process of natural evolution

• NO structural information required

• NO understanding of the mechanism required

Random Mutagenesis

• Generation of genetic diversity

• Screening and natural selection

Cont …..

Site-directed Mutagenesis

Or

Site Directed Mutagenesis is a powerful technique

where site specific changes in DNA sequence are produced in vitro-for instance to change an

amino acid residue into another by changing the codon sequence within

the gene sequence

Site Directed Mutagenesis is a molecular biology

technique in which a mutation is created at a

defined site in a DNA molecule known as a

plasmid. Wild-type gene sequence must be known.

CAG

GTC

CAG

+ primer

CAG

primer GCC

+ polymerase

CAG

GCC

replication

CAG

GTC

Wild type

translation

Val

MutantWild type protein

CGG

GCC

translation

Thr

Mutant protein

Only one amino acid changed

Val → Thr

(1)

(2)

(3)

(4)

(5)

(6)

Smith (1993)

Site directed Vs Random mutagenesis

-> site-directed mutagenesis

-> point mutations in particular known area

-> random mutagenesis

-> point mutations in all areas within DNA of interest

INVENTIONSite Directed Mutagenesis using oligonucleotide

was first described in 1978 by Michael Smith &

shared Nobel Prize in chemistry in October 1993

with Kary B. Mullis who developed the PCR

technique.

Site –directed mutagenesis

Requirements:

-> Knowledge of sequence and preferable Structure

(active site,….)

-> Understanding of mechanism

(knowledge about structure – function relationship)

Site Directed Mutagenesis

Cassette mutagenesis

Oligonucleotide directed mutagenesis

Using M13 DNA

Using Plasmid DNA

3. PCR amplified Oligonucleotide directed mutagenesis

4. Random mutagenesis

With Degenerate Oligonucleotide primers or

Using Nucleotide Analogues

CASSETTE MUTAGENESIS

Cleavage by a Restriction

Enzyme (RE) at a particular site

in the plasmid.

Ligation of an Oligonucleotide

containing Mutation in the gene

of interest to the plasmid.

RE that cuts at the plasmid

and Oligonucleotide is same,

permitting sticky ends of the

plasmid & inserts to ligate to

one another.

Site-directed mutagenesis –Oligonucleotide - directed method

Synthetic single-stranded fragments of DNA used for themutated clones.

In order to work, the primers must meet the followingcriteria:

-must contain desired mutation.

-mutation should be in the middle of the primer.

-the GC content should be at a minimum of 40% and shouldterminate in one or more of C or G bases.

Oligonucleotides are…

Oligonucleotide directed

mutagenesis

Basic Methods

1. Base pair substitution: Change

of one nucleotide (A-> C)

2. Insertion: Gaining one

additional nucleotide

3. Deletion: Loss of one nucleotide

Deletion Mutagenesis

1. Synthesize an Oligonucleotide

containing the changed sequence.

Ex.---ATT---Wild type sequence

(Codon for ILe)

---CTT---Desired Change (Leu)

---GAA---Mutagenic

Oligonucleotide (MO)

2.Hybridize MO ss form of

gene cloned into M13.

3.Synthesize second strand of

DNA with KLENOW fragment

&dNTPs.

4.Seal nick in new strand

with T4 DNA ligase.

5.Introduce into E. Coli.

6.ss + phage isolated from

plaques & screened by

hybridization.

Oligonucleotide directed

mutagenesis with M13 DNA:

Oligonucleotide

Directed

Mutagenesis

Using Plasmids

More common

Increases the chances of

obtaining the desired

mutation in the target

gene

Antibiotic resistance gene

restored and a second one

eliminated

Uses of Site Directed Mutagenesis

1.Site Directed Mutagenesis is also used to ‘engineer’commercially important Proteins for many differentpurposes, for example

•Improve stability

•Change specificity

•Reduce toxicity

Site Directed Mutagenesis enabled new approaches to drug designing –particularly in order to improve

FUNCTION.

Random Mutagenesis is used to construct large & diverse clone libraries of mutated

DNA fragments.

DNA sequencing

DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule.

It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA.

Applications of DNA sequencing

Knowledge of DNA sequences has

became valuable

for basic biological research

virology

medical diagnosisbiological

systematicsforensic biology

DNA sequencing helps to

Molecular biology:

to study genomes and the proteins they encode.

allows researchers to identify changes in genes

identify potential drug targets.

Evolutionary biology:

to study how different organisms are related and how they evolved.

DNA sequencing helps to

Metagenomics: The field of metagenomics identification of organisms present in a body of water, sewage, dirt, debris filtered from the air, or swab samples from organisms. Sequencing enables researchers to determine which types of microbes may be present in a microbiome.

Sanger Sequencing/ chain termination method

The most popular method for doing this is called the dideoxy method or Sanger method (named after its inventor, Frederick Sanger, who was awarded Nobel prize in chemistry in 1980[his second] for this achievment).

The Procedure

The DNA to be sequenced is prepared as a single strand.

This template DNA is supplied with

a mixture of all four normal (deoxy) nucleotides in ample quantities

dATP ,dGTP ,dCTP ,dTTP.

a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color:

ddATP ,ddGTP ,ddCTP ,ddTTP

DNA polymerase I

Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide instead of the normal deoxynucleotide.

At the end of the incubation period, the fragments are separated by length from longest to shortest. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.

Other methods

• Maxam-Gilbert sequencing

• Advanced methods and de novo sequencing

• Shotgun sequencing

• Bridge PCR

Referenceswww.bx.psu.edu/~ross/workmg/Isolat_analyz_genes_Chpt3.htm

www.bioinfo.org.cn/book/biochemistry/chapt28/sim1.htm

www.clfs.umd.edu/grad/mlfsc/res/RecombomamtDNA&Medicine.ppt

www.authorstream.com/.../mritunjaymtj-1389017-recombinant-dna-tech

http://www.rvc.ac.uk/Extranet/DNA_1/DNA_1_intro.htm

http://library.thinkquest.org/24355/data/light/details/media/recombinantanim.html

http://www.organoninc.com/products/consumer/

https://www.neb.com/applications/cloning.../site-directed-mutagenesis

www.idtdna.com/pages/.../01/.../methods-for-site-directed-mutagenesis

https://en.wikipedia.org/wiki/DNA_sequencing

https://www.dnalc.org/.../15479-Sanger-method-of-DNA-sequencing

www.genomenewsnetwork.org/resources/whats_a.../Chp2_2.shtm