Introduction to Genetic Analysis 9/e

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14 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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Gene Isolation and Manipulation

2

Recombinant DNA Technologies &
Genetic Engineering

The ability to isolate (clone) genes and manipulate
genes led to the birth of the new field of genetic
engineering.

3

Recombinant DNA Technologies &
Genetic Engineering

1.
Important factors

1.
Ability to fragment the genome.

1.
Restriction enzymes


2.
Ability to fuse two different DNA molecules.

1.
Complementary base pairing

2.
DNA ligase


1.
Ability to amplify a DNA region or gene.

1.
DNA polymerases

2.
Reverse transcriptase



Ability to zoom in on a specific DNA region or gene.


Molecular Probes

Create fragments of genomic DNA that can be inserted into a
vector by the use of restriction enzymes


Ligate these fragments with the vector DNA


Make many copies of the recombinant DNA


Analyze the fragments on an agarose gel


Making a genomic library

Steps in making recombinant DNA

Restriction
enzymes

Recognize and bind specific sequences on the genome (sequence specific).


Different restriction enzymes recognize and bind different target sequences.



EcoRI



GAATTC



CTTAAG




BamH1

-

GGATCC


CCTAGG



The target sequence is usually a
palindrome
(both strands have the same DNA sequence which
occurs as an inverted repeat).



5’ GAATTC 3’



3’ CTTAAG 5’


Cuts the sugar phosphate backbone


generate a number of smaller fragments from a large molecule.

6

Restriction Enzymes:

Different enzymes have different recognition sites

7

Restriction Enzymes


Cleave the DNA double helix at the sugar
-
phosphate backbone at
specific sequences.

Cohesive(sticky)
end

A hybrid or chimeric DNA molecule (human DNA + Bacterial plasmid
vector) is formed by complementary base pairing through the cohesive ends
of the two molecules.

G
AATTC

CTTAA
G

3’

GAATTC

CTTAAG

5’


AATTC


G

5’

3


GAATTC

CTTAAG

5’

3’

G
AATTC

CTTAA
G

G

CTTAA

5’

3


Cohesive(
sticky)
end

Figure 20
-
2

Formation of a recombinant DNA molecule

DNA molecules of two different
origins (human and
bacterial)


Joined together


Through complementary base
pairing


of cohesive ends generated by
restriction digestion


And ligation by the enzyme
DNA ligase

Inserting a gene into a recombinant DNA plasmid

Figure 20
-
3

Making Many Copies of the
Recombinant DNA Clones:

1.
Delivery of recombinant clone into host.


2.
Amplification of recombinant clone in host.


3.
Selection for recombinant clones.

Modes of delivering recombinant DNA into bacterial cells

Figure 20
-
8

Making a lot of cloned DNA

Figure 20
-
4

Different vectors can be used. They can be of bacterial, phage
and yeast origin. They differ in the length of DNA inserts they
can accept.




Polymerase chain reaction (PCR): Another
way to make more DNA from a small amount

Polymerase chain reaction

Figure 20
-
14


http://highered.mcgraw
-
hill.com/olc/dl/120078/micro15.swf

Sequencing of DNA


Using the Sanger Dideoxy
-
method

The structure of 2

,3

-
dideoxynucleotides

Figure 20
-
15

The dideoxy sequencing method

Figure 20
-
16a

http://smcg.cifn.unam.mx/enp
-
unam/03
-
EstructuraDelGenoma/animaciones/secuencia.swf

The dideoxy sequencing method

Figure 20
-
16b

The dideoxy sequencing method

Figure 20
-
16c

Reading the DNA sequence from an automatic sequencer

Figure 20
-
17

30

Genomic DNA Library Construction

1. Subject genomic DNA to partial restriction digests
and clone the fragments

31

Construction of
cDNA Libraries

A comprehensive collection of DNA
copies (cDNA) of the mRNAs
expressed in a specific cell.

35

cDNA Libraries:

Each clone is a DNA copy of one of the mRNAs in the cell

Summary


DNA cloning involves the cutting of DNA with restriction enzymes
and inserting the resulting DNA fragments into bacterial plasmid
vectors.


PCR (Polymerase chain reaction) allows the amplification of DNA
from tiny amounts of starting materials.


“Sanger” DNA sequencing uses di
-
deoxy(DD)
-
nucleotides. When they
are built into the growing chain, the chain will terminate. If ddATP is
used, one knows that the sequence at the place where the synthesis
stopped was an A. Separate recations are done for the other dd
-
nucleotides.


To make a library of all the transcripts in a tissue, the RNAs are first
made into complementary DNA (cDNA)