Restriction enzymes and site-specific DNA

cleavage
 An enzymatic technique for cleaving DNA molecules at specific
sites

 The enzymes are called restriction endonucleases or

restriction enzymes (RE) – technically known as type II
restriction endonucleases– five types identified
 Eg. BamHI, EcoRI, HindIII, HaeII, TaqI

 The nucleotide sequence recognized for cleavage by a restriction

enzyme is called the recognition sequence and the site of
cleavage is the restriction site

 Asymmetric cleavage vs symmetric cleavage

 Sticky ends vs blunt ends

 Sequences for restriction sites are virtually always palindromic

AGB 558: PLANT BIOTECHNOLOGY 1
Restriction enzymes and site-specific DNA
cleavage

AGB 558: PLANT BIOTECHNOLOGY 2

Restriction enzymes and site-specific DNA
cleavage

AGB 558: PLANT BIOTECHNOLOGY 3

Restriction enzymes and site-specific DNA
cleavage

AGB 558: PLANT BIOTECHNOLOGY 4

 Restriction enzymes-- Isoschizomers
 Isoschizomers are RE with the same recognition sequence

 Neoschizomers are subsets of Isoschizomers – RE with the

same recognition sequence but cleave at different restriction
sites Eg. SmaI and XmaI; TaiI and MaeII

https://www.neb.com AGB 558: PLANT BIOTECHNOLOGY 5

 Restriction enzymes-- Nomenclature

https://en.wikipedia.org/wiki/Restriction_enzyme#Nomenclature

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 The Molecular Unity of Life
 All creatures on earth share many features of the genetic
apparatus, including genetic information encoded in the
sequence of bases of DNA

 All creatures also share certain characteristics in their

biochemistry, including many enzymes and other proteins

 Why do organisms share a common set of similar genes

and proteins?

 The process of evolution takes place when a population of

organisms descended from a common ancestor gradually
changes in genetic composition through time – descent
with modification

AGB 558: PLANT BIOTECHNOLOGY 7

 Genomes and Proteomes
Size of
Total Genome # protein- % repetitive
Ploidy & # sequenced
Species Genome sequencing coding DNA
chrom. genome
size (Mbp) strategy genes sequence
(Mbp)
Rice
Whole genome
( Oryza
2n=24 shotgun and ~35% of the
sativa) ~400 ~370 37544
n=12, diploid Map-based or genome
hierarchical

>75% of the
Maize 2n=20 Hierarchical
~2500 ~2300 32,000 sequenced
(Zea mays) n=10 method
genome
Sequencing
Wheat 2n=6x=42
isolated >80% of the
(Triticum x=7, ~17,000 All 124,201
chromosome genome
aestivum) allohexaploid
arms
whole-genome
Barley 2n=2x=14 ~84% of the
shotgun
(Hordeum x=7, ~5100 ~4900 79,379 genome
seguencing,
vulgare) Diploid
POPSEQ
Sorghum 2n = 20 whole-genome
~61% of the
(Sorghum n=10 ~730 shotgun 678.9 34,496
genome
bicolor) Diploid sequencing
AGB 558: PLANT BIOTECHNOLOGY 8
Sanger DNA Sequencing

AGB 558: PLANT BIOTECHNOLOGY 9

Deoxy versus dideoxy nucleotides

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 Nucleosides
Base linked to a 2-deoxy-D-ribose at 1’ carbon

Nucleotides
• Nucleosides with a phosphate at 5’ carbon

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 Phosphodiester Bond
• DNA Polymerase

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 Dideoxy (Sanger) Method
4 Steps:
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Gel electrophoresis

AGB 558: PLANT BIOTECHNOLOGY 13

 Dideoxy (Sanger) Method
 ddNTP- 2’,3’-
dideoxynucleotide

 No 3’ hydroxyl

 Terminates chain when dNTPs

incorporated

 Add enough so each

ddNTP is randomly and
completely incorporated
at each base

AGB 558: PLANT BIOTECHNOLOGY 14

 Dideoxy Method
 Run four separate
reactions each with
different ddNTPs

 Run on a gel in four

separate lanes

 Read the gel from

the bottom up

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AGB 558: PLANT BIOTECHNOLOGY 19
 So What’s Wrong With It?
The dideoxy method is good only for 500-
750 bp reactions

Expensive

Takes a while

The human genome is about 3 billion bp

AGB 558: PLANT BIOTECHNOLOGY 20

Genetic variation and DNA Markers

Genetic differences among individuals

Types of DNA markers present in

gDNA

Applications of DNA markers

AGB 558: PLANT BIOTECHNOLOGY 21

 Genetic differences among individuals
 Differences in DNA sequences among individuals in a given species

 The human genome is ~3 billion bp organized into 23 distinct

chromosomes
 Protein coding gene sequences account for ~1.3%
 98.7% do not code for proteins
 All humans are 99.9% identical in DNA sequences
 Only 0.1% (3 million bp) differs from one human to the other except
for identical twins

 Some of these differences are associated with inherited differences in

biological traits

 Yet, many of these differences have no detectable effects on phenotype

 They can only be studied through direct examination of DNA itself –
they serve as genetic markers

AGB 558: PLANT BIOTECHNOLOGY 22

 Genetic differences among individuals
 A genetic marker is any difference in DNA, no matter how it is detected,
whose pattern of transmission from generation to generation can be
tracked

 Each individual who carries the marker also carries a length of

chromosome (DNA) on either side of it
 They mark a particular region of the genome – landmarks in chromosomes –
like signposts along highways

 Any difference in DNA sequence between two individuals can serve as a

genetic marker
 Genetic markers that are detected by direct analysis of DNA are called DNA
markers

 DNA markers of allow position of genes controlling biological traits of

agricultural importance to be located on chromosomes (genome mapping)
and their DNA isolated, identified, and studied

AGB 558: PLANT BIOTECHNOLOGY 23

 Genetic differences among individuals

How do we manipulate genomic DNA to reveal

genetic differences among individuals? The answer
to question is the focus of this lecture

The manipulation of DNA is the basic experimental

operation in modern genetics

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