WSSP-14 Chapter 3Analyzing DNA –Restriction Digests

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p. 1-10

Cloning cDNA fragments into pTripleEX2

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Restriction enzymes are used for cloning and analyzing DNA fragments

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1 2 3 41 2 3 4

ONs

1 2 3 4

MPs

4.0

4.0

1.0

1.0

2.01.6

1.0

0.5

3.04.0

M 1 2 3 4

RI/HindIII

2.01.6

1.0

0.5

3.04.0

M 1 2 3 4

PstI

1 2 3 41 2 3 4

ONs

1 2 3 4

MPs

3.0

4.0

2.0

1.0

2.01.6

1.0

0.5

3.0

4.0

M 1 2 3 4

RI/HindIII

Bacteriophage - "eaters of bacteria"

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Restriction enzymes - endonucleases, Cleave a specific DNA sequence

Protect bacteria from phage infection, digest phage DNA after infection

p. 3-1

GAATTCCTTAAG

GCTTAA

AATTC G

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Why don't the restriction enzymes degrade the cell DNA?

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Cellular DNA protected by methylases -

block restriction enzyme activity

p. 3-1

GAATTCCTTAAG

GCTTAA

AATTC G

GAATTCCTTAAG

M

MMethylase

GAATTCCTTAAG

M

M

EcoRI EcoRI

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DNA-binding proteins make non-covalent contacts to the DNA

Methyl group

CH3

Methylation blocks binding by the protein

NOTE: DNA Methylation in bacteria has a different purpose then DNA methylation in eukaryotesVertebrate CpG methylation – reduces gene expression

in adult somatic cells, hypermethylation silences genes

Genomic imprinting, X-chromosome inactivation, suppression of repetitive elements

Bacterial Dam methylase (GATC) involved in mismatch repair, replication, and gene expression

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Each strain of bacteria has a specific set of restriction

enzymes • EcoRI from Escherichia coli • BamHI from Bacillus amyloliqueraciens• PvuI and PvuII are different enzymes

from same strain.

•Originally purified by individual labs, Nathans, Smith

•Now supplied by companies - NEB, Promega

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Sequence Recognition and cleavage:

a) 5' overhang EcoRI GAATTC GOH pAATTC

CTTAAG CTTAAp OHG

b) 3' overhang KpnI GGTACCGGTACOH

pCCCATGG Cp

OHCATGG

c) Blunt end SmaI CCCGGG CCCOHpGGG

GGGCCC GGGpOHCCCp. 3-2© 2014 WSSP

DNA fragments with compatible sticky ends can be ligated togetherExample:

NNG AATTCNNN NNGAATTCNNNNNCTTAA GNNN NNCTTAAGNNN

Not

NNG GGCCCNNN NNGGGCCCNNNNNCTTAA GNNN NNCTTAAGNNN

Ligase+ATPOH P

P OH

OH P

P OH

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DNA fragments with blunt ends can be ligated together

Example:

NNGAA TTCNNN NNGAATTCNNNNNCTT AAGNNN NNCTTAAGNNN

NNGAA CCCNNN NNGAACCCNNNNNCTT GGGNNN NNCTTGGGNNN

Ligase

Ligase

OH P

OH P

OHP

OHP

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Sequence Recognition and cleavage: d) Degenerate:AvaII GGWCC: GGTCC, GGACC

AvaI CPyCGPuG CTCGAG Py stands for pyrimidine- T or C CTCGGG Pu stands for purine - A or G CCCGAG

CCCGGG

C TCGAG C CCGGG CCCGAGGAGCT C GGGCC C GAGCCC

DdeI CTNAG: CTAAG, CTGAG, CTCAG, CTTAG

BbsI cleaves GAAGACNNCTTCTGNNNNNN

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Recognition sites for the SfiI restriction enzyme

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p. 3-3© 2014 WSSP

Activity: in units which corresponds to a specified level of enzyme activity.

NEB defines a unit as:

“One unit of restriction endonuclease activity is defined as the amount of enzyme required to completely digest 1 g of substrate DNA in a total reaction volume of 0.05 ml in one hour using the NEB buffer provided.”

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Before setting up a restriction digest check to make sure that you are using the proper conditions!

Improper conditions can denature (inactivate) the enzyme or cause non-specific digestion (Star activity)

Activity of an enzyme can change under different conditions:pH- 7.5, 8.0, 8.5salt concentration- 20 mM, -150 mMdivalent cations- Mg++reducing reagent- DTT carrier protein-BSA temperature- 37C, RT, 60C

Restriction enzymes are proteins with optimal conditions

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p. 3-4

Information on Restriction Enzymes

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4. Set up the Reaction:

Add in the following order:

Sterile ddH2O 7.0 l10 X restriction buffer 2.0 lMiniprep DNA (0.5 g) 10.0 lEnzyme (20 U/l) 1.0 lTotal volume 20.0 l

The two most important rules in enzymes

• Always keep enzymes on ice or in a cooler.

• Always use a fresh tip when pipeting from the enzyme stocks.

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Set up a master mix multiple digests:

Single MultipleSterile ddH2O 7.0 l 35.0 l10 X restriction buffer 2.0 l 10.0 lMiniprep DNA (0.5 g) 10.0 l**none**Enzyme (20 U/l) 1.0 l 5.0 lTotal volume 20.0 l 10.0 l Mix aliquot

10.0 l DNA

p. 3-7

Never put anything into your yellow MP tube!!!

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5. Incubate reactions at the appropriate temperature for the appropriate time.

Usually 37˚C and incubate 1 hr or more.

6. If running on a gel: Add gel loading dyeEDTA - Stops reaction.Dyes (BPB and XC) - to help see sample while

loading and monitor electrophoresisGlycerol - so sample sits at bottom of the well

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Using a restriction digest to map a plasmid

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Map of the pTriplEX2 plasmid

p. 3-9

cDNA Fragment

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Why not test cut the plasmid with SfiI?

2. Can’t tell if single cut or non insert3. It is also expensive.

p. 3-10

1. May have lost the site in cloning

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Theoretical PvuII-HF digests of plasmids

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Why are there 3 bands in the uncut lane?

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A real Digest gel of clones from the Duckweed cDNA library

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5. Does your digest look complete?Are the intensities of the bands proportional to the size?

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Bands Generated from Partial Digests

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6. Is there a PvuII site in the insert?Are there more than two bands?

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6. Is there a PvuII site in the insert?Are there multiple bands?

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7. What is the size of the insert?

p. 3-13

700 bp - 700 bp = 0 bp

2.9 Kb

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8. Do the results from the digest and the PCR agree?

p. 3-14200 bp - 200 bp = 0 bp

700 bp - 700 bp = 0 bp

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8. Do the results from the 20JM1.10 digest and the PCR agree?

p. 3-14600 bp - 200 bp = 400 bp

1100 bp - 700 bp = 400 bp

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8. Do the results from the 20JM2.10 digest and the PCR agree?

p. 3-141700 bp - 200 bp = 1500 bp

2200 bp - 700 bp = 1500 bp

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8. How do we figure out the insert size from the 20JM4.10 digest?

p. 3-14

1200 bp + 400 bp-700 bp = 900 bp

2900 bp

1200 bp

400 bp

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8. Do the results from the 20JM4.10 digest and the PCR agree?

p. 3-141100 bp - 200 bp = 900 bp 1200 bp +400 bp-

700 bp = 900 bp

Enter the results of the RD gel and whether the clone should be sequenced into your school’s Google Docs Clone Report sheet

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Enter the conclusions into the Google Docs Clone report

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