Manipulating DNA

Manipulating DNA

• Scientists use their knowledge of the structure of DNA and its chemical properties to study and change DNA molecules

• Different techniques are used to study and change DNA molecules

• Genetic Engineering – making changes in the DNA code of a living organism

• Bacteria are the workhorses of modern biotechnology

Tools of Molecular Biology: Recombinant DNA

• Recombinant DNA techniques can help biologists produce large quantities of a desired protein

• To work with genes in the laboratory, biologists often use bacterial plasmids, small, circular DNA molecules

– Plasmids can:• can carry virtually any gene,• can act as vectors, DNA carriers that move genes from one

cell to another, and• are ideal for gene cloning, the production of multiple identical

copies of a gene-carrying piece of DNA.

Recombinant DNA

• Recombinant DNA is produced by combining two ingredients:• a bacterial plasmid and • the gene of interest.

• To combine these ingredients, a piece of DNA must be spliced into a plasmid

Creating Recombinant DNA

• In order to create Recombinant DNA, there needs to be:– DNA extraction

• Cells opened to separate DNA from other cell parts

– Cutting DNA

• DNA too large to study, so biologists “cut” them into smaller fragments using restriction enzymes. Many restriction enzymes are known and each one cuts DNA at a specific sequence of nucleotides

• Produces pieces of DNA called restriction fragments with “sticky ends” important for joining DNA from different sources.

– Splicing DNA back together

• DNA ligase connects the DNA pieces into continuous strands by forming bonds between adjacent nucleotides

Restriction EnzymesRecognition site (recognition sequence)for a restriction enzyme

Restrictionenzyme

Sticky end

Sticky end

A restriction enzyme cuts the DNA into fragments.

1

Restriction EnzymesRecognition site (recognition sequence)for a restriction enzyme

Restrictionenzyme

Sticky end

Sticky end

DNA

A DNA fragment is added from another source.

A restriction enzyme cuts the DNA into fragments.

1

2

Restriction EnzymesRecognition site (recognition sequence)for a restriction enzyme

Restrictionenzyme

Sticky end

Sticky end

DNA

A DNA fragment is added from another source.

A restriction enzyme cuts the DNA into fragments.

Fragments stick together bybase pairing.

1

2

3

Restriction EnzymesRecognition site (recognition sequence)for a restriction enzyme

Restrictionenzyme

Sticky end

Sticky end

DNA

DNAligase

Recombinant DNA molecule

A DNA fragment is added from another source.

A restriction enzyme cuts the DNA into fragments.

Fragments stick together bybase pairing.

DNA ligase joins the fragments into strands.

1

2

3

4

Restriction Enzymes

Recognition sequences

DNA sequence

Restriction enzyme EcoRI cuts the DNA into fragments. Sticky end

Restriction Enzymes

Recognition sequences

DNA sequence

Restriction enzyme EcoRI cuts the DNA into fragments. Sticky end

Finding the Gene of Interest

• How can a researcher obtain DNA that encodes a particular gene of interest? First, you have to have an idea of what the gene is you want to work with (get a genomic library). Then:

• Using a nucleic acid probe consisting of a short single strand of DNA with a complementary sequence and labeled with either a radioactive isotope or a fluorescent dye.

• Or by synthesizing it through reverse transcriptase (viral enzyme that makes DNA)

• Or by making it by scratch with machines

DNA Profiling and Forensic Science

• DNA profiling• can be used to determine if two samples of genetic

material are from a particular individual and• has rapidly revolutionized the field of forensics, the

scientific analysis of evidence from crime scenes.

– To produce a DNA profile, scientists compare sequences in the genome that vary from person to person.

DNA Profiling and Forensic Science

– DNA profiling can be used to• test the guilt of suspected criminals,• identify tissue samples of victims,• resolve paternity cases,• identify contraband animal products, and• trace the evolutionary history of organisms.

DNA Profiling Techniques: Making Copies of DNA

• Polymerase Chain Reaction (PCR) technique– Allows biologists to make many copies of a

specific piece of DNA– DNA strands separated with heat, then cooled

to allow DNA Polymerase to start making new copies of DNA

– A few dozen heat and cool cycles results in many copies of DNA

Making Copies of DNADNA polymerase adds complementary strand

DNA heated to separate strands

DNA fragment to be copied

PCRcycles 1

DNAcopies 1

2

2

3

4

4

8

5 etc.

16 etc.

DNA Profiling Techniques: STR Analysis

– Short tandem repeats (STRs) are:• short sequences of DNA that are repeated many times,

tandemly (one after another), in the genome.

– STR analysis• Proves two samples come from the same person - Everyone

has these repetitive DNA sequences, but in different lengths and a different number of them

• compares the lengths of STR sequences at specific sites in the genome

• uses gel electrophoresis, a method for sorting DNA by size

DNA Profiling Techniques: Gel Electrophoresis

• Gel electrophoresis– Used to separate DNA fragments. DNA

fragments placed in a gel and electricity is applied to the gel. DNA molecules are negatively charged and move towards the positive end of the gel. Smaller DNA fragments move faster and farther

– This technique used to compare the genomes of different organisms or even different people

Gel Electrophoresis

DNA plus restriction enzyme

Mixture of DNA fragments

Gel

Power source

Longer fragments

Shorter fragments

Gel Electrophoresis

Gel Electrophoresis

DNA from

Victim

DNA from

Suspect #1

DNA from

Suspect #2

DNA from

Suspect #3

DNA from

Suspect #4

Blood found at Crime Scene

Which suspect should have more questioning?