Cutting and Pasting DNA

• The cutters are called restriction enzymes, they cut DNA at specific nucleotide sequences

Restriction enzymecuts DNA intofragments

1

2

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4

Addition of a DNAfragment fromanother source

Fragments sticktogether bybase pairing

DNA ligasejoins the strands

Recognition sequence for a restriction enzyme

Sticky end

Sticky end

Recombinant DNA molecule

How Do I Obtain the Gene of Interest?

• The “shotgun” approach - millions of recombinant plasmids are produced; a genomic library

• How a DNA probe tags a gene

Radioactive probe (DNA)

Single-stranded DNA

Mix with single-stranded DNA from various bacterial clones

Base pairing indicatesthe gene of interest

• The reverse transcriptase method

Transcription1

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Introns removed and exons spliced together

Isolation of mRNA from cell and addition of reverse transcriptase

5 Synthesis of secondDNA strand

Synthesis of cDNAstrand

Exon Intron Exon Intron Exon

Cell nucleus

DNA ofeukaryoticgene

RNAtranscript

mRNA

Cytoplasm

Reversetranscriptase

Test tube

cDNA strandbeing synthesized

cDNA ofgene with-out introns

• DNA technology revolutionized forensics

WHO ARE YOU?

– forensics is the scientific analysis ofevidence from crime scene investigations

• DNA fingerprinting has been available since 1986

• Used in CSI, victim IDs; Cheddar Man

Crimes and Other Investigations

• Polymerase Chain Reaction (PCR) copies DNA

DNA Fingerprinting Techniques - PCR

• DNA amplification by PCR

Tinyamountof DNA

8421

Number of DNA molecules

• DNA fragments cut by restriction enzymes are seen as “bands” in an electrophoresis gel

Longerfragments

Shorterfragments

CrimesceneDNA

SuspectDNA

Defendant’sblood

Blood on defendant’sclothes

Victim’sblood

• The science of studying entire genomes

GENOMICS

• In 1990, an international consortiumbegan the Human Genome Project

The Human Genome Project

– sequencing the human genome

– challenges: large size, and the fact that only a small amount of our DNA is genetic

• Genome mapping

Chop up withrestriction enzyme

Chromosome

Sequencefragments

DNA fragments

Alignfragments

Reassemblefull sequence

• Gene therapy cures disease by altering human genes

HUMAN GENE THERAPY

Normal humangene isolatedand cloned

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Human geneinsertedinto virus

Virus injectedinto patient

Healthy individual

Nonharmfulvirus (vector)

Virus containingnormal human gene

Bone marrow

• Great power brings both promise and danger

SAFETY AND ETHICAL ISSUES

– strict laboratory safety procedures are used

– negotiators from 130 countries agreed on a Biosafety Protocol for GM foods