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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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