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Mani Lecture 2

A Molecular Genetics Toolbox I:

Tools to clone, amplify, analyze and sequence DNA

1.  Cold Spring Harbor Labs Learning Centre

2  Molecular Biology of the Gene (Watson et al.l 6th (International) Edition

3  8th Day of Creation. Horace Freeland Judson (Historical)

Insulin and sugar metabolism

Type-1 diabetes: Not enough insulin and high blood sugar

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How is insulin produced?

Once purified from animal tissue (beef pancreas).

Now: recombinant insulin.

Identify human gene(s) for insulin.

Make bacteria produce mRNA and protein

Purify the protein

Package, Store, Q/C, transport.

From previous lecture(s)

1.  The genome (all our chromosomal DNA) encodes more than 100,000 proteins.

2.  A small sequence of DNA encodes each protein.

DNA cloning, amplification and expression of selected DNA sequences (e.g DNA encoding human Insulin)

A key concept is of DNA hybridization, based on “Watson-Crick” base-pair interactions.

Perfectly base-paired helices Remain stable at higher temperatures

DNA cloning and in vivo amplification of DNA sequences

Key1: Restriction endonucleases (evolved for bacterial defense)

Key2: Plasmids (self replicating, extra chromosomal circles of DNA).

Key3: Bacterial Transformation and growth

Werner Arber, Daniel Nathans, Hamilton O. Smith

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!  Restriction Enzymes: site-specific cutting tools for DNA

Salvadore Luria

Lambda Phage

Strain A Strain B

Plaques No Plaques (occasional, rare plaque)

Phage plaque On E.coli lawn

Bacteriophage (e.g. Lambda) Infect and kill/lyse bacteria

!  Restriction Enzymes: site-specific cutting tools for DNA

Lambda Phage

Strain A Strain B

Plaques No Plaques (or occasional rare plaque)

Strain B: 1.  has a restriction endonuclease that cleaves Phage DNA (eg. EcoR1) at a specific 6-mer

2.  A methyl transferase that protects its own genome.

Thanks to an evolutionary arms race there are many different restriction enzymes

Salvadore Luria

•  Restriction enzymes recognize specific short palindromic sequences in DNA:

typically 4, 6 or 8 bp

The enzyme EcoRI recognizes and cuts GAATTC which occurs on average every ~4.1 kb

!  Restriction Enzymes: site-specific cutting tools for DNA

•  Restriction enzymes cut DNA into “manageable-sized” fragments

GAATTC CTTAAG

5’

5’ 3’

3’

5’ single-stranded cohesive ends

G AATTC CTTAA G

5’

5’ 3’

3’ 3’ 5’

5’ 3’

!  DNA cloning: amplifying DNA sequences in vivo

Chromosome-sized DNA (50 Mb) 1. Extract and

purify DNA

2. Cut DNA into gene-sized fragments using a restriction enzyme e.g. EcoRI

Insert genomic fragments into a suitable cloning vector

2X 106 kb: We need to “purify” and grow the ~ 2kb we are interested in.

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!  Plasmid cloning vectors (viral – e.g. lambda-based vectors can also be used)

Bacteria have plasmids or “episomes” (additional chromosomes) with genes conferring antibiotic resistance.

Bacteria and phage also have DNA ligases that stick together/ repair broken DNA

Bacteria have restriction enzymes that cut DNA at specific sequences

!  Plasmid cloning vectors

Plasmids: small, circular, typically containing the following features:

•  Origin of replication: directs plasmid replication

•  Selectable marker: antibiotic resistance gene

Vector pUC19

Size: 2.8 kb

pUC19 is a general-purpose cloning vector that accepts DNA inserts of up to 10 kb

The lacZ region allows ‘blue-white screening’ using the stain X-Gal

White colonies contain the vector with an insert that disrupts lacZ’

Blue colonies contain the original vector (no inserts)

Location of restriction enzyme sites for inserting DNA

•  Cloning site: a collection of unique restriction enzyme sites

!  Creating recombinant DNA molecules: ligation of vector and genomic fragments

Cohesive ends of vector anneal with those of random genomic DNA fragment

Genomic DNA: EcoRI fragments

EcoRI Plasmid vector ……pUC19

Linearized vector DNA

T4 DNA ligase seals the gap in the sugar-phosphate backbone ‘Recombinant DNA’

E. coli cells washed in CaCl2 can take up DNA and be transformed by it

!  Introduction of recombinant molecules into E. coli (transformation)

v Extract plasmid DNAs from a colony

To check for cloned DNA cut with EcoRI

Clone Number

Cells are placed on agar medium containing an antibiotic e.g. ampicillin

Individual transformed cells grow to produce visible colonies

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!  Genomic libraries: collections of clones corresponding to all sequences in a genome

How to identify the colony containing insulin coding DNA?

Human 1000,000 kb genome. From a plasmid library each plasmid housing a 10kb fragment

100,000 colonies corresponds to “one genome equivalent”

(32P DNA probe for insulin DNA)

Expression vector

E coli transform and induce

Purify Insulin

Screen Library

Isolate recombinant insulin in pure form ready for human use (is a little more complicated)

Step 2 : E. coli strain 1 produce and purify Chain 1 E coli strain 2 produce and purify Chain 2

Step 3: Mix chains under conditions where appropriate disulfide bonds form

Step 1: Clone appropriate constructs

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2. PCR: in vitro amplification of DNA sequences

!  DNA can be chemically synthesized: oligonucleotides (oligos)

•  Custom-designed oligos can be ordered on-line from many biotech companies

• (Eg. http://www.sigmaaldrich.com/life-science/custom-oligos.html)

•  Short (21 – 29 base) single-stranded oligonucleotide of defined base sequence

Synthetic oligonucleotide primers were key to the development of:

(a) PCR and

(b) DNA sequencing

!  The availability of oligos (1980s) made the invention of PCR possible

•  Kary Mullis (Nobel Prize 1993) proposed that a pair of oligos could be used to prime the amplification of a target DNA sequence in a cyclical manner

3’

5’

5’

3’

1. Separate the DNA strands by heating to 98oC

2. Reduce temp to allow oligo primers to anneal (base pair)

Oligo Primer 1 (forward)

3’ 5’

Oligo Primer 2 (reverse)

5’ 3’

3. Reduce temp; Add dNTPs and DNA polymerase

3’

3’ 5’

5’

Double stranded genomic DNA

Steps 1 – 3 represent a synthesis cycle Repeat many times

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the habitat for Thermus aquaticus.Morning Glory Pool: Yellowstone •  PCR uses a heat-stable DNA polymerase from the thermophilic bacterium

Thermus aquaticus (Taq DNA pol)

•  This enabled automation

PCR machine

A typical PCR amplification cycle

!  Amplification proceeds exponentially

After 35 cycle there will theoretically be 236 = 68 billion copies of the target sequence

1 billion bases ~ 1 picogram (10-12)

68 billion of 2000 base fragment = 1.4 micrograms if starting from one template molecule.

35 cycles

Detection of Biological Agents Used for Terrorism: Are We Ready ...by M Kellogg - 201019 Nov 2009... as a portable PCR machine that reports a quantitative value. ... after the First Gulf War because the rapid tests used in that war were ...www.clinchem.org/cgi/content/full/56/1/10 - Similar

1. Chagas outbreaks in his home country inspired Núñez-Mujica to find a cheap, portable PCR machine to help diagnose those infected with Chagas (trypansome)

BioTechniques 24 Aug 2010: Cheap PCR: new low cost machines challenge..

2. Rapid Diagnosis rRT- PCR on-site diagnosis for animal pathogens/. E.g. Portable PCR cycler machines are used to test cattle for TB

PCR and Variations of the basic technique are used for “Diagnostics.”

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Design forward and reverse primers for the gene of interest

PCR product

AA BB AB Gel electrophoresis of PCR products digested with EcoR1 from different individuals

!  Major application: genotyping of alleles based on a Restriction Fragment Length Polymorphism (RFLP)

The alleles differ by a Single Nucleotide Polymorphism (SNP)

Homozygotes: AA, BB Heterozygote: AB

Allele A contains an EcoRI restriction site

EcoRI GAATTC

Allele B lacks the EcoRI site due to a single base mutation

GGATTC

!  Genotyping based on allele-specific length differences

•  Amplification of alleles that show length polymorphisms:

•  Microsatellite sequences are one type of VNTR e.g. (CA)n

•  Widely dispersed throughout the human genome

Allele A (CA)3

Allele B

(CA)8

AA AB BB

Agarose gel showing PCR products

Homozygotes: AA, BB Heterozygote: AB

•  Variable Number Tandem Repeats (VNTRs)

!  PCR can be used to amplify several VNTR loci simultaneously

VNTR Locus 1

VNTR Locus 2

VNTR Locus 3

M 1 2 3 4 5 6 M 7 8 9 10 11 12M

Multiplex genotyping using fluorescently labelled primers designed to amplify alleles of different sizes: these can be separated on the same gel

Multiplexing allows alleles at many different loci to be investigated simultaneously

Major applications in 1) Forensic science 2) Paternity testing 3) Population Genetics 4) Evolutionary genetics

✧  Colin Pitchfork: First person convicted using DNA Forensics.

✧  Norborough Village Murder ( Lynda Mann, 1983, Dawn Ashworth, 1986)

Alec Jefferies (1985)

http://www.dailymail.co.uk/news/article-2079782/ Stephen-Lawrence-trial-Gary-Dobson-David-Norris-guilty-murder.html