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Flashcards in Biotechnology 😄 Deck (12):
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Used to deduce original sequence of DNA

Dideoxynucleotides halt DNA polymerization at each base, generating sequences of various lengths that encompass the entire sequence

Sanger DNA sequencing

1

Molecular biology lab procedure that is used to synthesize many copies of a desired fragment of DNA

Steps (repeated multiple times)
1. DNA is denatured to generate 2 separate
2. During cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified
3. Half-stable DNA polymerase replicates the DNA sequence following each primer

Polymerase chain reaction

2

A DNA sample is electrophoresed on a gel and then transferred to a filter

The filter is then soaked in a denaturant and subsequently exposed to a labelled DNA prove that recognizes and anneals to the complementary strand

The resulting double stranded labeled piece of DNA is visualized when the filter is exposed to film

Used to determine which restriction fragments of DNA are associated with a particular gene

Southern blot (DNA sample)

3

Similar technique, except that Northern blotting involves radioactive DNA prove binding to sample RNA

Used to measure sizes and amounts of specific mRNA molecules to answer questions about gene expression

Northern blot (RNA sample)

4

Sample protein is separated via gel electrophoresis and transferred to a filter

Labelled antibody is used to bind to relevant protein

Used to measure amount of antigen (proteins) or antibody

Western blot (Protein sample)

5

Thousands of Nucleic acid sequences are arranged in grids on glass or silicon

DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amounts of complementary binding

Microarrays (color coded)

6

Enzyme-linked immunosorbent assay

A rapid immunologic technique testing for antigen-antibody reactivity

Patient's blood is probed with either:
- test antigen (coupled to color-generating enzyme) to see if immune system recognizes it
- test antibody (coupled to color-generating enzyme) to see if a certain antigen is present

Intense color reaction: (+) result

ELISA

7



Fluorescent probe binds to specific gene site of interest

Specific localization of genes and direct visualization of anomalies (e.g. microdeletions) at molecular level

FISH (Fluorescence in situ hybridization)

8

Inherited difference in the pattern of restriction

Important in understanding various single-gene and multigenic diseases

Useful as a diagnostic tool for diseases involving single-base changes, or deletions/insertions of DNA into a restriction fragment

Restriction Fragment Length Polymorphism

9

Is the production of recombinant DNA molecules that is self-perpetuating

Tissue mRNA is isolated and exposed to reverse transcriptase, forming a cDNA (lacks introns) library

Cloning

10

Steps of cloning methods

DNA fragments are inserted into bacterial plasmids that contain antibiotic resistance genes. These plasmids can be selected for using media containing antibiotic and amplified

Restriction enzymes cleave DNA at 4-6 base pairs palindromic sequences, allowing for insertion of a fragment into a plasmid

11

Treatment option for diseases caused by deficiency of a gene product

The strategy is to clone a gene into a vector that will readily be taken up and incorporated into the genome of a host cell

Gene therapy