Download MBBS Biochemistry PPT 29 Recombinant Dna Technology Lecture Notes

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RECOMBINANT DNA

TECHNOLOGY

1

Objectives

? Recombinant DNA

? Probes

? Restriction map

? Gene cloning

? Gene library

? Cloning vectors

? RFLP

? DNA Fingerprinting

? DNA foot printing

? Genomic imprinting

2
Three techniques

that facilitate

analysis of human DNA

3

Steps of Genetic Engineering

? Cutting DNA at a specific site
? Joining of two DNA fragments to create a

novel DNA

? Cloning or amplification of available DNA
? Expression of a DNA to obtain its product
? Sequencing of a DNA molecule
? Synthesis of an oligonucleotide

4
Application of Recombinant

Technology

? Understanding of diseases: Sickle cell

anaemia, Thalassemia

? Diagnosis of diseases: AIDS, Hepatitis
? Treatment of Diseases: Human Insulin
? Prevention of diseases: Hepatitis vaccines
? Gene therapy: SCID

5

Recombinant DNA

Two DNA fragments of interest: Even from different source or species

Cohesive or sticky ends with complementary sequences

Treated with same RE

in some cases blunt ends are joined by

Homopolymer tailing

A small synthetic duplex oligonucleotide having RE sites attached

5` ends of the linker DNA are phosphorylated by polynucleotide kinase

6
Different Enzymes used in DNA Recombinant

technology Enzyme

Functions

DNA ligase

joins of ends

DNA Pol I

Synthesis of double

Restriction

stranded DNA

Endonuclease

DNAse I

Produces nicks in

sDNA

Exonuclease I I

Removes nt from 3`

end

exonuclease

Removes from 5` end

Polynucleotide kinase Phosphorylates 5` OH

group

Alk phosphatase

Removes 5` PO4

S1 nuclease

Degrades sDNA

7

Recognition sequence of

restriction endonuclease

EcoRI shows

two-fold rotational symmetry

8
Nomenclature of

Restriction endonuclease

Why they are named so

What are sticky ends and

blunt ends

Restriction sites frequency

9

TaqI and Hae I I I

(Haemophilus aegyptius)

restriction endonucleases

10
Restriction Map

involves the size analysis of restriction fragments

produced by several restriction enzymes

individually and in combination

11

12
Probe

15-20 nt long oligont used to search a particular DNA fragment

Chemically synthesized DNA or RNA pieces

Generally labelled with radioactive material or a fluoroscent label

Construction of a probe

From Genetic database

homologous gene in other species : heterologous gene probe

mRNA

protein----- rich in Trp and Met

13

Label ing of a Probe

End labelling: 32P

Random labelling: During synthesis: Usually GTP:

Fluorescent labelled

14
End-labelling of a gene probe at the 5' end with

alkaline phosphatase and polynucleotide kinase

15

End-labelling of a gene probe at the 3'

end using terminal transferase

16
Cloning

Process of producing large number of identical copies from

one single original DNA molecule or fragment

Importance

To study gene: purified form and in sufficient amount

To study sequencing, expression in different tissues

under different conditions

Methods of amplification

Cell based

Cell free

17

Outline of gene cloning

18
19

Gene Library

? All of the DNA extracted from an organism

and digested with a restriction enzyme will

result in a collection of clones. This collection

of clones is known as a gene library

20
Type of gene library

Genomic library: Total chromosomal DNA of organism

cDNA library: represents the mRNA from a cell or tissue

at a specific point in time

Type of gene library depends on final application of DNA

Goal: Production of new or modified proteins or determination

of tissue specific expression and timing pattern

cDNA library

Goal: To understand the control of protein production for a

particular gene

Genomic library

21

Gene Library

? 1. Construction of gene library

? Digesting genomic DNA molecules

? Choice of Enzyme?

? Ligating DNA molecules

? Carried out at 10o C to lower the kinetic energy

and to reduce the chances of sticky ends

parting

? 2. Cloning vectors

? 3. Screening Gene Library

22
Numbers of clones required for representation of DNA

in a genome library

23

Ligation molecules with cohesive ends. Complementary cohesive ends base -pair,

forming a temporary link between two DNA fragments. This association of fragments

is stabilised by the formation of 3' to 5' phosphodiester linkages between cohesive ends,

a reaction catalysed by DNA ligase.

24
Comparison of general steps in the construction of

genomic and cDNA library

25

Cloning Vector

. DNA elements that may be stably maintained

and propagated in a host organism for which

the vector has replication functions.

. Host organism is a bacterium such as E. coli
. vector with a replication origin in E. coli will

replicate (together with any incorporated
DNA) efficiently

26
Comparison of vectors general y available for cloning

Vector

Host cell

Vector structure Insert

range(kb)

M13

E coli

Circular virus

1-4

Plasmid

E coli

Circular plasmid

1-5

Phage

E coli

Linear virus

2-25

Cosmids

E coli

Circular plasmid

35-45

BACs

E coli

Circular plasmid

50-300

YACs

S.

Linear

100-2000

Cerevisiae chromosome

27

Structure of E.Coli plasmid cloning vector pBR322

28
Replica plating to detect recombinant

plasmids

29

Map and important features of pUC18

30
Principle of blue/white selection for

the detection of recombinant vectors.

31

Cosmid vectors

Cosmid vectors incorporate the cos sites from phage l and also

the essential features of a plasmid, such as the plasmid origin

of replication, a gene for drug resistance, and several unique

restriction sites for insertion of the DNA to be cloned.

32
Expression vector

The inserted sequence must be placed so that

its reading frame is in phase with the regulatory sequence

33

Shuttle vectors

? Shuttle vectors have origins of replication for

yeast and bacteria such as E. coli. This means

that constructs may be prepared rapidly in the

bacteria and delivered into yeast for

expression studies.

34
Delivery of vectors into Eukaryotes

? Transfection:

? to deliver recombinant molecules into animal cel s

? 1. making the membrane permeable with

divalent cations / use of polyethylene glycol (PEG)

? 2. electroporation: subjected to pulses of a high-

voltage gradient

? 3. lipofection: DNA is encapsulated by a core of

lipid-coated particles

35

SCREENING GENE LIBRARIES

36
Colony hybridisation technique

for locating specific bacterial

colonies harbouring recombinant

plasmid vectors containing

desired DNA fragments.

Application of gene cloning

Molecular analysis of disease

Normal gene variation--Polymorphism

Gene variation causing disease--Beta globin gene

Detection of point mutation---Sickle cell disease

Detection of deletion /insertion/rearrangement--

Beta globin gene

Prenatal diagnosis

Preimplantation diagnosis: done in IVF

Disease linkage analysis--Microsatel ite repeats in families

Forensic medicine

38
Structural Alterations of the a-Globin Gene

39

Schematic representation of the -globin gene cluster and

of the lesions in some genetic disorders.

40
Sickle cel disease

41

Pedigree analysis of Sickle cel disease

42
Restriction Fragment Length Polymorphism and SNPs

major use of SNPs/RFLPs is in the definition of inherited diseases

in which the functional deficit is unknown

SNPs/RFLPs can be used to establish linkage groups, which in turn,

by the process of chromosome walking,

will eventually define the disease locus

43

The technique of chromosome walking

44
Microsatellite repeat variation in some diseases

Disease

Repeat

Normal length

Mutation length

of repeats

Fragile X

(CGG)n

6-54

200-1000

syndrome

Fredriech ataxia (GAA)n

7-22

7200

Myotonic

(CTG)n

50-35

50-4000

dystrophy

Spino cerebel ar (CAG)n

19-36

43-81

ataxia

45

? DNA Finger printing

46
? Alec Jeffrey in 1984

? Each individual has unique sequences

47

48
49

50
51

Purpose

? 1. Paternity dispute
? 2. Criminal identification

52
Method

? Isolation of DNA
? Digestion of DNA by RE
? Amplification
? Separation by gel electrophoresis
? Blotting
? Hybridisation with radiolabelled probe
? Autoradiography

53

54
55

? DNA Footprinting

56
Footprinting with DNase I

? Footprinting enables a control region to be

positioned within a restriction fragment that has

been identified by gel retardation.

? Footprinting works on the basis that the

interaction with a regulatory protein protects the

DNA in the region of a control sequence from the

degradative action of an endonuclease such as

deoxyribonuclease (DNase) I.

? This phenomenon can be used to locate the

protein binding site on the DNA molecule.

57

A bound protein can protect a

region of DNA that is much longer

than the control sequence.

58
59

Genomic imprinting

60
What Mendel found out

? Two parents make equal contribution to the

character

? The effect of an allele is independent of

whether it comes from the female or male

gamete

61

62
63

64
65

66
67

68
69

Summary

? Recombinent DNA is the joining of two fragments cut with same restriction

endonuclease

? Probe is a 15-20 nt long oligonucleotide used to search a particular DNA fragment

? Cloning is a process of producing large number of identical copies from one single

original DNA molecule or fragment

? Type of gene library depends on final application of DNA

? Delivery of vector in bacteria is cal ed transformation and in animal cel s is

transfection

? RFLP is a technique used to identify the individual as wel as to detect disease

condition such as Sickle cel anaemia

? DNA finger printing is to identify the individual based on differential tandem

repeat sequences

? Footprinting works on the basis that the interaction with a regulatory protein

protects the DNA from the degradative action of an endonuclease such as

deoxyribonuclease (DNase) I.

? Genomic imprinting usual y uses DNA methylation (epigenetic regulati on).

70

This post was last modified on 05 April 2022