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BIOLOGY - CLUTCH
CH.20 - BIOTECHNOLOGY
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
EXAMPLE:
● Restriction endonuclease – cleaves specific, palindromic sequence of DNA
□ Sticky ends – overhang left after cleavage capable of H-bonding with complementary sequence
BIOLOGY - CLUTCH
CH.20 - BIOTECHNOLOGY
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CONCEPT: DNA CLONING
● Complementary DNA (cDNA) – double stranded DNA synthesized from mRNA
□ Reverse transcriptase – forms cDNA from an RNA template EXAMPLE:
● 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
EXAMPLE:
BIOLOGY - CLUTCH
CH.20 - BIOTECHNOLOGY
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CONCEPT: DNA CLONING
● Bacteria are transformed with the new plasmids, some are transformed with plasmids that did not get the gene of interest.
EXAMPLE:
● Antibiotic resistance and color changes are frequently used to separate the transformed bacteria. EXAMPLE:
BIOLOGY - CLUTCH
CH.20 - BIOTECHNOLOGY
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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
EXAMPLE:
● PCR requires a few ingredients:
□ Deoxynucleotide triphosphates (dNTPs)
□ Sequence specific primers
□ DNA polymerase (usually Taq polymerase)
□ Starter DNA
EXAMPLE:
BIOLOGY - CLUTCH
CH.20 - BIOTECHNOLOGY
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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
EXAMPLE:
2. Annealing – the mixture is cooled allowing the primer to hybridize to the strand
EXAMPLE:
3. Elongation – the temperature is set to the optimal operating temperature of the polymerase
EXAMPLE:
BIOLOGY - CLUTCH
CH.20 - BIOTECHNOLOGY
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CONCEPT: DNA SEQUENCING
● Dideoxy DNA sequencing – an early sequencing method that relied of the use of dideoxynucleotide triphosphates
EXAMPLE:
● ddNTPs terminate elongation of the strand because there is no available 3’ –OH group to continue the polymerization.
EXAMPLE:
BIOLOGY - CLUTCH
CH.20 - BIOTECHNOLOGY
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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
EXAMPLE:
● 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
EXAMPLE:
BIOLOGY - CLUTCH
CH.20 - BIOTECHNOLOGY
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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
EXAMPLE:
● 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
EXAMPLE:
BIOLOGY - CLUTCH
CH.20 - BIOTECHNOLOGY
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