Chapter 12 DNA Technology and Genomics

I. Recombinant DNA Technology

1. Biotechnology is the manipulation of organisms or their components to make useful products by recombinant DNA technology (genetic engineering, molecular cloning, or gene cloning).  In 1973, Stanley Cohen and Herbert Boyer used a plasmid to clone DNA, i.e., performed the first cloning experiment.

2. Recombinant DNA techniques

(1)  Plasmid is a small circular DNA that can self-replicate in bacteria and yeasts.

(2)  Plasmids can carry any gene and are key tools for gene cloning (molecular cloning).  A clone means a group of identical cells generated from a single ancestral cell.

(3)  Vector is a plasmid to serve as a gene carrier during molecular cloning.  Insert is a DNA fragment that includes the gene of interest.

(4)  A restriction enzyme, also called a molecular scissors, is a bacterial enzyme that cut DNA at a specific palindrome (hair-pin, or stem-loop) sequence.  For example, EcoRI recognizes and cut the DNA sequence 5'-GAATTC-3'.

(5)  A DNA ligase, also called a molecular glue, is a bacterial enzyme that paste DNA into continuous strands.

(6)  Genomic library is a set of DNA fragments (carried by plasmids or phages) that represent an organism's entire genome.

(7)  Complementary DNA (cDNA) is a DNA molecule made by mRNA as a template and reverse transcriptase.

(8)  Nucleic acid probe is a radioactively, enzymatically, or fluorescently labeled single-stranded nucleic acid molecule used to screen a specific gene or other nucleotide sequence within a mass of DNA.  Automatic DNA synthesizer is a machine to produce a customized, single-stranded DNA (DNA primer, oligo DNA, oligonucleotide).

(9) A powerful new technique is the CRISPR-Cas9 system for gene (genome) editing. Cas9 protein can cut DNA molecules directed by a guide RNA molecule as a homing device.

3. Genetically modified organism (GMO)

(1)  A GMO is an organism that gets one or more artificial modified genes.  If the newly obtained gene is from another organism, we call this a transgenic organism.

(2)  Humulin^® is human insulin produced by genetically modified bacteria.  Insulin is a protein hormone made by the pancreas to decrease the level of glucose in the blood.

(3)  Pharm (Pharmaceutical; pronounce like Farm) animals represent transgenic animals carrying a recombinant human gene.

4. Gene therapy is a treatment for a disease in which the patient's defective gene is supplemented or altered.

II. DNA Profiling

1. DNA profiling (also called DNA testing, DNA typing, or genetic fingerprinting) is the analysis of DNA samples in criminal investigations to provide evidence to forensic science.

2. Polymerase chain reaction (PCR) is a technique used to make many copies of a small amount of specific segment of DNA.  A DNA template is mixed with a pair of primers, the thermostable Taq DNA polymerase and other ingredients.  The solution is then exposed to cycles of heating (to separate the DNA strands) and cooling (to allow double-stranded DAN to reform) in a PCR machine.

3. Gel electrophoresis is a technique for sorting macromolecules by size, for instance, DNA, RNA and proteins.

4. Genetic marker is an allele tracked in a genetic study or a specific section of DNA that contains a particular allele.

5. The most often used genetic markers are inherited variations in the lengths of repetitive DNA segments, consisting of nucleotide sequences that are present in multiple copies in the genome.

6. Repetitive DNA contains a series of repeats called short tandem repeats (STRs).  Short tandem repeat (STR) analysis is a method of DNA profiling that compares the number of STR segments at specific sites in the genome.

7. In 1995, DNA profiling was the first use in the O. J. Simpson murder trial.

8. Subsequently, DNA profiling was applied in cases of President Bill Clinton, the World Trade Center attack on September 11, 2001, paternity, protection of endangered species and archeology.

9. Single nucleotide polymorphism (SNP, pronounced snip) is a one-nucleotide variation in DNA sequence found within the genomes of at least 1% of a population.

10. Restriction fragment length polymorphism (RFLP, pronounced rif-lip) is a variation in the length of a restriction fragment (i.e., restriction fragment analysis).  RFLP can be produced when a homologous DNA sequence containing SNPs that alter the DNA sequence recognized by a restriction enzyme.

III. Genomics

1. Genomics is the scientific study of whole genomes (complete sets of genes and their interactions).

2. Human genome project (HGP) is an international collaborative effort to map and sequence the DNA of the entire human genome.  This project was begun in 1990 and completed in 2003.

3. The human genome contains approximately 3.0 billion (2.9 x 10⁹) base pairs deposited in GenBank, National Institutes of Health (NIH).

4. The biggest surprise from the HGP is the small number of human genes.  The current estimate is about 21,000 genes which are very close to the number of nematode worm (20,100 genes).

5. Proteomics is the scientific study of the full set of proteins encoded by a genome.  The number of different proteins in human is over 100,000 proteins (versus about 21,000 genes).