Chapter 14 GENETIC TECHNOLOGY

A. Manipulation and Modification of DNA 1. Restriction Enzymes Recognize specific sequences of DNA (usually palindromes) and make cuts. These cuts usually produce “sticky ends”

Used to remove segments of DNA which can be “pasted” into another piece of DNA using the enzyme ligase.

2. Reverse Transcriptase An enzyme produced by retroviruses Creates a double stranded DNA molecule from an RNA template Complementary DNA (cDNA) is produced when reverse transcriptase creates DNA from purified mRNA cDNA lacks introns

3. Vectors Molecules that maintain a DNA molecule of interest and carry it into a cell Plasmids (extra pieces of DNA found in bacteria) and retroviruses are common vectors.

B. DNA Amplification 1. Polymerase chain reaction (PCR) Used to produce millions of copies of a target DNA sequence within hours Requires the following items which are subjected to cycles of heating and cooling: A target DNA sequence Primers DNA nucleotides Heat stable DNA polymerase (Taq)

C. Separation and Analysis of DNA 1. Gel electrophoresis DNA fragments of various sizes are loaded into an agarose gel where an electrical current is applied. DNA has a negative charge and migrates towards a positive charge. Smaller DNA fragments are able to move faster than larger fragments. After staining, a banding pattern is visible.

2. DNA Fingerprints (basically southern blot) Patterns produced when DNA is cut by restriction enzymes Produced by looking at: SNPs (single nucleotide polymorphisms) - highly variable regions of genomes which vary from one individual to the next SNPs can be cut with restriction enzymes which will produce different sized DNA fragments in different individuals.

Comparison of repeated DNA sequences (variable number tandem repeat=short tandem repeat=minisatellite= 미소부체 ) Certain areas in the chromosomes have repeated DNA sequences. The number of these sequences varies from one individual to the next. Cutting with restriction enzymes will produce different sized fragments depending on the number of repeats.

12 Southern blot

3. Hybridization Used to detect a particular DNA segment from multiple bands within a gel In Southern blotting, DNA bands can be transferred from a gel to a nylon membrane and then exposed to a probe. Specific probes will base pair to the DNA if interest (=hybridization)

DNA strands are antiparallel. 5’ to 3’ strand 3’ to 5’ strand Numbering of strands is based on position of deoxyribose sugars.

16 Linking the Fragments ] DNA ligase forms a phosphodiester bond between 3’-hydroxyl of the growing strand and the 5’-phosphate of an Okazaki fragment

17 ddNTP

18 Figure Sanger’s Sequencing Method

D. DNA sequencing The technique used for reading the sequence of a DNA molecule The Sanger Method (ddNTP)

Sequencing on DNA microarrays

E. Applications of Recombinant DNA technology Transgenic organisms Carry foreign genes

Gene therapy Replacement of a nonfunctioning gene in somatic cells in an attempt to fix an inborn genetic error. Monitoring gene expression DNA chips can be used to monitor gene expression in different cell types or under different conditions.

F. Gene Silencing Blocking the expression of a particular gene to look for missing functions that correspond to that gene. Antisense technology blocks mRNA which prevents protein synthesis Knockout technology uses homologous recombination to swap a disabled form of a gene for a natural form of the gene

plasmid strain