!! www.clutchprep.com
CONCEPT: DNA CLONING DNA cloning is a technique that inserts a foreign gene into a living host to replicate the gene and produce gene products. Transformation the process by which an organism takes foreign DNA and incorporates it into its genome Plasmid a small DNA molecule autonomous of the bacterial chromosome that serves as a cloning vector Restriction endonuclease cleaves specific, palindromic sequence of DNA Sticky ends overhang left after cleavage capable of H-bonding with complementary sequence Page 2
CONCEPT: DNA CLONING Complementary DNA (cdna) double stranded DNA synthesized from mrna Reverse transcriptase forms cdna from an RNA template Restriction endonucleases are used to open up the plasmid to insert the gene of interest. DNA ligase seals the new gene into the plasmid Recombinant DNA DNA that is formed through laboratory methods and combines genetic material from multiple sources Page 3
CONCEPT: DNA CLONING Bacteria are transformed with the new plasmids, some are transformed with plasmids that did not get the gene of interest. Antibiotic resistance and color changes are frequently used to separate the transformed bacteria. Page 4
CONCEPT: POLYMERASE CHAIN REACTION Polymerase chain reaction (PCR) is a technique used to amplify as little as one piece of DNA, generating many copies. Thermal cycler the piece of lab equipment that carries out PCR PCR requires a few ingredients: Deoxynucleotide triphosphates (dntps) Sequence specific primers DNA polymerase (usually Taq polymerase) Starter DNA Page 5
CONCEPT: POLYMERASE CHAIN REACTION The process has three repeated steps: 1. Denaturation the mixture is heated and the double stranded DNA separates into single strands 2. Annealing the mixture is cooled allowing the primer to hybridize to the strand 3. Elongation the temperature is set to the optimal operating temperature of the polymerase Page 6
CONCEPT: DNA SEQUENCING Dideoxy DNA sequencing an early sequencing method that relied of the use of dideoxynucleotide triphosphates ddntps terminate elongation of the strand because there is no available 3 OH group to continue the polymerization. Page 7
CONCEPT: DNA FINGERPRINTING Genetic map an ordered list of gene loci along a chromosome Linkage map a genetic map based on recombination frequency Physical map a genetic map based on the physical locations of the genes along the chromosome Genetic marker genes or DNA sequences that have a known location and are easily identifiable Polymorphic more than one phenotype is associated with a gene Single-nucleotide polymorphisms (SNPs) DNA sequence between alleles varies by a single base pair Page 8
CONCEPT: DNA FINGERPRINTING Genetic profile analysis of the genetic markers unique to an individual Short tandem repeats (microsatellites) short repeated sequences of DNA that vary between alleles and individuals Gel electrophoresis a method for separating macromolecules based on size and charge Southern blot a method used to detect specific DNA sequences Northern blot a method used to detect specific RNA sequences Western blot a method used to detect specific proteins Page 9