producing DNA fragments

what do many human diseases result from

the inability to produce metabollic chemicals such as proteins (insulins)

what does recombuniant DNA technology allow?

genes to be manipulated, altered and transffered from organism to organism

what does the genetic code process include?

transcription and translation which is universal

stages to make protein using DNA technology

1. isolation of the DNA fragments that have the gene for the desired protein

2. insertion of the DNA fragment into a vector

3. transformation, the transfer of the DNA into suitable host cells

4. identification of host cells that have successfully taken up the gene markers

5. growth/cloning of a population of host cells

usage of reverse transcriptase to isolate a gene

1. beta cells specialised in insulin production make lot’s of mRNA that codes insulin

2. mRNA acts as a template on which a single-stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase

3. single stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme

4. double stranded DNA is formed on the template of the template of the cDNA using DNA polymerase

what are restriction endonucleases

bacteria defend themselves by producing enzymes that cut up the viral DNA. the enzymes are restriction endonucleases.

using restriction endonucleases

• DNA is cut into recognition sequences. a cut is made between two opposite pairs leaves ‘blunt ends’

• other cut in a staggered fashion to expose a DNA palindrome and create sticky ends

what is another way of isolating DNA fragments

the gene machine

what is the ‘gene machine’ and how does it work

• the amino acid sequence of a protein is determined, from which the mRNA codons and DNA triplets can subsequently be worked out

• computer designs a series of small, overlapping single strands of nucleotides called oligonucleotides

• a double stranded copy of this gene is generated, and using sticky ends is inserted into a plasmid vector

• the oligonucleotides are then joined together to make a gene that has no introns. the gene is then replicated using the polymerase chain reaction

• a double stranded copy of this gene is generated and using sticky ends is inserted into a plasmid vector

• genes are checked using sequencing techniques and those errors are rejected

advantages of the gene machine

• any sequence can be produced

• very accurate

• intron free

• prokaryotes can be used