exAM 3 DNA tech.

What is DNA technology?

Techniques used to isolate, purify, analyze, and manipulate DNA sequences. Includes genetic engineering, genomics, and bioinformatics.

What is a GMO?

A genetically modified organism — an organism whose genome has been engineered to introduce or change a genetically determined trait.

What is biotechnology?

Any technique used to modify a biological system. It includes genomics (the study of genomes) and bioinformatics (math and computer science applied to biological data).

What is genomics?

The study of an organism's entire genome.

What is bioinformatics?

The application of mathematics and computer science to analyze biological data, particularly genomic sequences.

What are restriction enzymes (endonucleases)?

Enzymes that cut DNA at specific recognition sequences called restriction sites, producing restriction fragments. They are used to join DNA molecules from different sources.

What are sticky ends?

Single-stranded overhangs of DNA left after a restriction enzyme cuts. They can base-pair with complementary sticky ends from another fragment, allowing different DNA pieces to be joined.

What is gene cloning?

The process of making multiple copies of a gene and inserting it into a host cell (usually a bacterium) for amplification.

What is a cloning vector (plasmid)?

A small, circular DNA molecule used to carry and insert a DNA fragment of interest into a host cell for cloning, creating a recombinant bacterium (a GMO).

What are the steps of gene cloning using a plasmid?

1) Cut the target DNA and the plasmid with the same restriction enzyme (creating matching sticky ends). 2) Mix the cut DNA and cut plasmid with DNA ligase to seal them together. 3) Reintroduce the recombinant plasmid into bacteria via transformation.

What is transformation in gene cloning?

The process by which a plasmid is reintroduced into a bacterial host cell so the bacteria can express the inserted gene.

What does PCR stand for, and what is it used for?

Polymerase Chain Reaction. It is used to amplify (make many copies of) a specific DNA sequence exponentially.

What are the three steps of PCR and their temperatures?

1) Denaturation (~96°C): double-stranded DNA is separated into single strands. 2) Annealing (~55-65°C): primers bind to their complementary sequences at each end of the target. 3) Extension (~72°C): DNA polymerase replicates the target sequence, producing 2 copies.

What is the result of multiple rounds of PCR?

Exponential amplification — a few rounds produce tens of thousands of copies of the target gene.

What is gel electrophoresis?

A technique that separates DNA fragments by size using an electric current through an agarose gel.

Why does DNA move toward the positive end in gel electrophoresis?

Because DNA has a negative charge, it is attracted to the positive electrode (opposites attract).

In gel electrophoresis, which fragments travel further — larger or smaller?

Smaller fragments travel further through the gel because they move more easily. Larger fragments travel a shorter distance.

What is a DNA marker ladder in gel electrophoresis?

A standard control containing DNA fragments of known sizes, used to compare and estimate the sizes of unknown fragments by how far they travel.

What is agarose gel electrophoresis used for in biotechnology?

To compare PCR results, analyze restriction fragments, and perform DNA fingerprinting by comparing fragment sizes against a known standard ladder.

What is the general process for making insulin using gene cloning?

1) Cut a bacterial plasmid with a restriction enzyme to create sticky ends. 2) Isolate the human insulin gene using PCR. 3) Insert the insulin gene (with matching sticky ends) into the plasmid using DNA ligase. 4) Transform E. coli with the recombinant plasmid. 5) Select bacteria that took up the plasmid. 6) Grow selected bacteria to produce insulin.

How are bacteria that successfully took up the recombinant plasmid selected in the insulin example?

The plasmid contains an ampicillin resistance gene. Bacteria are grown with ampicillin. Those that did NOT take up the plasmid grow blue; those that DID take up the plasmid grow clear. Blue colonies are removed.

What is cDNA (complementary DNA)?

DNA synthesized from an mRNA template using reverse transcriptase. It is complementary to the mRNA and lacks introns, making it useful for cloning eukaryotic genes into bacteria.

What is reverse transcriptase, and why is it important for making cDNA?

Reverse transcriptase is an enzyme that synthesizes DNA from an RNA template — the reverse of the normal central dogma (DNA → RNA → Protein). It is used to create cDNA from mRNA.

Why is cDNA preferred over genomic DNA when cloning eukaryotic genes into bacteria?

cDNA is made from processed mRNA (introns already removed), so it contains only the coding sequence. Bacteria cannot splice out introns, so cDNA must be used.

What are stem cells?

Undifferentiated precursor cells that have the potential to develop into different specialized cell types.

What does totipotent mean?

A cell that can develop into a fully functioning organism (e.g., a fertilized egg — the very first cell after fertilization).

What does pluripotent mean?

A cell that has the potential to develop into nearly every cell type in the body (e.g., embryonic stem cells).

What does multipotent mean?

A cell that can develop into only a limited number of cell types (e.g., adult stem cells).

What is a knockout mouse?

A mouse in which a specific gene has been made nonfunctional by introducing a nonfunctional version (transgene) into embryonic stem cells. Two transgenic mice (Gg × Gg) are bred to produce homozygous recessive (gg) knockout offspring.

What is a transgene?

A gene taken from one organism's genome and inserted into another organism's genome through genetic engineering.

What is a transgenic organism?

An organism that contains a transgene from a genetic engineering process.

What is recombinant DNA?

Modified DNA created by combining DNA sequences from different sources (e.g., inserting a human gene into a bacterial plasmid).

What does CRISPR stand for?

Clustered Regularly Interspaced Short Palindromic Repeats.

What is Cas9?

A DNA endonuclease (enzyme that cleaves phosphodiester bonds in DNA) guided by RNA to a specific target sequence where it makes a precise cut. It acts as molecular scissors.

How does CRISPR-Cas9 work?

The CRISPR-Cas9 molecule contains a guide RNA complementary to the target gene. Once the guide RNA matches the target, Cas9 cleaves the DNA at that site. A new gene can then be inserted or the sequence can be repaired by DNA polymerase.

What is the PAM sequence, and why is it important for CRISPR-Cas9?

PAM (Protospacer Adjacent Motif) is a specific DNA sequence adjacent to the target site that Cas9 must recognize before it can bind and unzip the DNA to match the guide RNA. Each Cas9 is specific to its target gene.

What is gene therapy?

The introduction of a healthy gene into a cell line to correct a genetic disorder.

What is somatic gene therapy?

Gene therapy targeting adult body (somatic) cells. Cells are cultured, transformed with an expression vector containing the therapeutic transgene, and reintroduced to the body. This is permitted in humans.

What is germline gene therapy?

Gene therapy where the corrective gene is introduced into germline cells, making the change heritable and passed to future offspring. This is NOT allowed in humans because it leads to "designer babies," which is considered unethical.

What is DNA fingerprinting (DNA profiling)?

A technique used to identify individuals by examining their unique DNA sequence, using PCR and gel electrophoresis for analysis.

What are Short Tandem Repeats (STRs)?

Short DNA sequences (2-6 base pairs long) repeated in tandem. The number of repeats at each locus varies among individuals, making STRs useful for unique identification.

How many STR loci do forensic scientists examine, and why is that sufficient?

Forensic scientists examine 13 STR loci. The probability of two people having the same repeat count at all 13 loci is essentially zero.

What are the steps of DNA fingerprinting?

1) Cut DNA with restriction enzymes. 2) Run the cut STR fragments on gel electrophoresis. 3) Analyze and compare the differences in fragment band patterns between individuals.

What are RFLPs (Restriction Fragment Length Polymorphisms)?

DNA fragments of different lengths generated from the same genomic region using restriction enzymes. Differences in fragment length reflect differences in DNA sequence. Analyzed by agarose gel electrophoresis.

How are RFLPs used in medicine?

Restriction sites serve as markers to indicate whether someone has inherited a genomic region associated with a disease, helping to assess genetic disease risk.

What is a SNP (Single-Nucleotide Polymorphism)?

A single base pair variation at a specific position in the genome. SNPs are a key source of human genetic variation, and certain SNPs are associated with increased disease risk (e.g., sickle cell anemia involves a SNP).

What is the difference between an RFLP and a SNP?

A SNP is a variation at a single nucleotide. An RFLP refers to differences in the lengths of restriction fragments between individuals. SNPs can create or destroy restriction sites, which can cause RFLPs.

What is an expression vector?

A vector (similar to a plasmid) used in somatic gene therapy that contains a target transgene, allowing it to be expressed in the host cell after transformation.