Recombinant DNA Technology for the non-

science major.

Historical Perspective

• Pre-history before 1928

• Ancient before 1944

• Medieval 1944-1952

• Renaissance 1953-1971

• Modern Era 1971 to present

Further Historical Perspective

Geneticists have known for a long time how to isolate DNA from cells.

Geneticists have known for a long time how to chop DNA into small pieces.

What geneticists did not know how to do until the early 1970s was to replicate small fragments of DNA.

1970s Breakthrough:

• The discovery of the restriction enzyme (or restriction endonuclease).

Properties of RE:

• Cut double-stranded DNA at specific target sites.

• Allow fragments of DNA that have been cut with the same RE to be rejoined.

Properties of RE con’t:

• There are hundreds of popular RE. They all recognize a small target sequence (4-8 b.p.).

The joining of two DNA fragments by DNA ligase produces a recombinant DNA molecule

Therefore, eukaryotic DNA could be propagated in prokaryotic cells.

A great breakthrough!!!!!

•

Carriers of foreign DNA are Vectors:

• Most vectors are derived from:• 1. Plasmids • 2. Bacteriophages• 3. Cosmids (artificial

constructions)

• A prokaryotic vector should:

• 1. Be capable of autonomous replication independent of the main bacterial chromosome

• 2. Be easy to isolate, i.e. small.

• 3. Be non-toxic to host cells.

• 4. Have space for foreign inserts.

• 5. Have unique restriction sites for common restriction enzymes.

• 6. Have convenient markers for selection of transformants, e.g. antibiotic resistance genes.

• 7. Be relaxed, i.e. multiple copies in a host cell.

• A bacteriophage is a virus that infects a bacteria.

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Introduction to PCR

• PCR (polymerase chain reaction).

* p r c

What is PCR?

• PCR is site-specific in vitro DNA replication.

DNA Replication Review:

• Add DNA polymerase, all 4 DNA building blocks ???

5’ CTGACGCTGCTGCATGCTAGCT 3’

3’ GACTACGACGACGTACGATCGA 5’

DNA Replication Review:

• Primers are required:

5’ CTGACGCTGCTGCATGCTAGCT 3’

CGA 5’

5’ CTG

3’ GACTACGACGACGTACGATCGA 5’

DNA Replication Review:

• Primers are required:

5’ CTGACGCTGCTGCATGCTAGCT 3’

. . . t a c g a t CGA 5’

5’ CTG a t g c t g . . . .

3’ GACTACGACGACGTACGATCGA 5’

Action of DNA Polymerases:

• DNA polymerases can add new nucleotides to an exposed 3’ end!

PCR reaction mix:

• All 4 DNA building blocks (A,C, G, & T)

• Taq DNA polymerase (heat resistant)

• DNA to be replicated

• A pair of primers

Human Chromosome # 8: | | | | | |

| || || || | add primers: | |

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Human Chromosome

# 8:

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PCR product?

• The binding of the primers determines where the DNA is replicated.

• The PCR product is a double-stranded DNA molecule with its ends defined by the location of primer binding sites.

PCR

• PCR reaction mix incubates for about 2 hours in a thermocycler (fancy incubator).

• The thermocycler heats and cools through 30-40 temperatures cycles.

• DNALC: PCR Animation

Two impt. PCR questions:

• What is the source of a pair of primers?

What information is a prerequisite for PCR?

PCR Applications:

#1- Cell-free rapid gene cloning!!

#2- Gene cloning/amplification from a miniscule sample size.

Introduction to Agarose Gel Electrophoresis

Weigh out ~ a gram of agarose.

• Mix the agarose with 50- 100 ml of buffer.

• Heat to dissolve the agarose.

• Assemble the gel tray and comb.

Pour the gel.

• Pick up the DNA sample with a microliter pipettor.

• Load one DNA sample into each well on the gel.

Connect the gel to a low voltage power supply.

After completion of the run, add a DNA staining material and visualize the DNA

under UV light.

• Analyze the results.

• Gel Electrophoresis

• Electrophoresis

Introduction to DNA Sequencing

Technology

Dideoxy Sequencing (Sanger Sequencing, Chain Terminator

method).• Clone the fragments to be sequenced

into the virus M13.

• Why M13?

• The clones that are isolated are single-stranded DNA.

M13

AGCATGATGTCGAGCGAGTCGTACGGT-----^^^

Primer

DNA sequencing reaction:1) DNA fragment to be sequenced cloned into the vector

M13

2) DNA polymerase

3) “Universal” primer

4) All 4 DNA building blocks

5) One ddNTP

The most popular technique is based on the dideoxynucleotide.

Set up 4 separate reactions. Each reaction contians one of the 4 ddNTPs. Each ddNTP is tagged with a radioactive tracer.

A reaction (with ddA) 21, 26, 29, . . . .T reaction (with ddT) 25, 31, 35, . . . . .C reaction (with ddC) 22, 23, 27, . . . . G reaction (with ddG) ??

M13

AGCATGATGTCGAGCGAGTCGTACGGT-----^^^

Primer (20 nt.)

• Each reaction generates a set of unique fragment lengths.

• All fragment lengths are represented.

• None of the fragments are present in more than one reaction.

• DNA sequencing technology requires gel electrophoresis system with the ability to separate DNA fragments that separate by one b.p.

DNA sequencing, as performed in the 1980s (manually) is slow

and labor intensive.

• NCBI HomePage

• DNALC: Cycle Sequencing

• CE Theory

• A Timeline of The Human Genome

• YEAR# human genes mapped to a definite chromosome location# years it would take to sequence the human

genome• 1967 none   sequencing not possible yet

• 1977 3 genes mapped 

• 4,000,000 years to finish at 1977 rate

• 198712 genes mapped • 1000 years to finish at 1987 rate

1997 30,000 genes mapped • 50 years to finish at present rate

• Organisms sequenced

• Year # genomes sequenced • 1994 0• 1995 2• 1996 4• 1997 8 (est.)• 199830 (est.)• 2001 ~75

• DOE Joint Genome Institute