Gene Therapy Overview
Summary of Gene Therapy
- Created by: emma louise
- Created on: 06-05-14 20:51
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- DNA Technology
- In Vivo Cloning
- Sticky ends will join to other complementary sticky ends
- DNA ligase used to join phosphate sugar backbone
- Fragment and vector cut with same restriction endonuclease
- Sticky ends allow binding
- Transformation (plasmid taken up by vector)
- Mixed in a medium containing calcium ions; bacteria becomes permeable
- Gene Markers
- Antibiotic Resistance
- Inserted gene takes the place of one resistance and keeps another
- Fluorescent Markers
- Cells that have taken up the gene will glow
- Enzyme Markers
- Lactase gene, turns substrate blue
- Antibiotic Resistance
- In Vitro Cloning
- Polymerase Chain Reaction
- 1) 95 degrees celcius; strands separate
- 2) 55 degrees celcius; DNA primers added- prevents stands rejoining
- 3) 72 degrees celcius; DNA polymerase join nucleotides - two new strands formed
- Copies DNA fragments, rapid and efficient
- Polymerase Chain Reaction
- Recombinant DNA
- To increase yield from animals or crop
- To improve nutrient content of food
- To introduce resistance
- To make crop plants tolerant to herbicides
- To make vaccines
- Locating and Sequencing Genes
- DNA Probes
- DNA probe made of bases complementary to DNA sequence
- DNA Sequencing
- 1) Four test tubes set up, each with one terminator nucleotide base, primer,mixture of nucleotides and DNA fragments to be sequenced
- 2) Random binding of nucleotides will eventually cause fragments to terminate after every base, creating different size fragments
- Gel Electrophresis
- 1) Fragments placed in wells in agar gel
- 2) Voltage applied
- 3) Resistance of gel means smaller molecules move faster
- As smaller fragments move further, when sequencing read from bottom to top
- Restriction Mapping
- Cutting DNA with a series of different restriction enzymes
- Fragments produced separated by gel electrophresis
- This can now be done automatically by a computer
- DNA Probes
- Medical Diagnosis
- Can be used to screen for specific genes
- Can be used to identify carriers of genes
- Producing DNA Fragments
- Using Reverse Transcriptase
- Using Retroviruses- able to synthesise DNA from RNA using reverse transcriptase
- 1) isolate
- 2) Relevant mRNA extracted
- 3) Reverse transcriptase makes DNA from mRNA - DNA known as cDNA
- Known as cDNA as it is made up of nucleotides complementary to mRNA
- To make the other stand DNA polymerase builds up complementary nucleotides
- Using Restriction Endonucleases
- Cuts DNA at specific recognition sites
- Some leave sticky ends - staggered edge; exposed unpaired bases
- Using Reverse Transcriptase
- In Vivo Cloning
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