Chapter 20 - Recombinant DNA Technology

molecular cloning

production of large quantities of identical copies of a specific DNA molecule

research on structure and organization of DNA

study gene expression

study protein products to understand their structure/function

producing important commercial products from the protein encoded by a gene

identical copies of DNA molecules can be manipulated for what reasons

recombinant DNA (rDNA)

genetic material from multiple sources are joined together to create unique sequences not otherwise found in the host genome

medicinal proteins (insulin, etc.), vaccines, diagnostic testing, gene therapy, DNA fingerprinting, and agriculture (GMOs)

why is recombinant DNA useful

number of short tandem repeats an individual has varies → they can be amplified via PCR and separated via gel electrophoresis to match an unknown sample to a known one

how is recombinant DNA used for fingerprinting

comes from Bacillus thuringiensis bacterial species and produced Cry proteins that are toxic to some insect species

what is the Bt gene

Cry proteins

what protein can corn be genetically modified to express that’s used as a safe microbial insecticide

pest caterpillars

what is Cry protein GMO corn particularly used to control

1) isolate the DNA segment to be cloned

2) select a cloning vector

3) Join DNA fragments together

4) Clone rDNA into host organism

4 basic steps of molecular cloning

bacteria produce them as a defense mechanism against bacteriophage infection

what produces restriction enzymes and for what reason

they restrict or prevent viral infection by degrading the DNA of invading viruses

what do restriction enzymes do when bacteria use them as defense mechanism

they recognize and bind to DNA at a specific nucleotide sequence (restriction sites) and cut both strands of the DNA by cleaving the phosphodiester backbone (digestion)

how are restriction enzymes involved in isolation o DNA segment for cloning

palindrome

what type of structure is the restriction site often

reads the same both strands of DNA when read in the 5’ to 3’ direction

what is a palindrome

allows the enzyme to recognize the sequence no matter which direction it approaches the DNA

benefit of the restriction site being a palindrome

1) blunt end (no complementary end for base pairing)

2) cohesive end (single stranded 5’ or 3’ overhang)

what are the 2 possible outcomes of cleavage at the restriction site

-complementary ends of DNA are not necessary

-less effective

-about 50% oriented wrong way

-can ligate fragments digested by different sequences/RE

pros and cons of blunt end cleavage

-complementary ends of DNA are necessary

-more effective

-able to orientate

pros and cons of sticky end cleavage

the RNA

for expressed regions where can DNA be isolated from

DNA copies which are made from mRNA molecules isolated from cultured cells of tissue samples → only represent genes that are being expressed in the cells at the time the library was made

what do complementary DNA libraries contain

all of the DNA, coding and noncoding, in a genome

what does genomic DNA contain

reverse transcription

what process is used to produce cDNA

-enzymes uses mRNA as template and forms double stranded mRNA/cDNA duplex

-then mRNA is partially digested with RNAse H to produce gaps in the strand

-remaining 3’ ends of the remaining mRNA serve as primers for DNA pol I which synthesizes the second DNA strand

-results in double stranded cDNA molecules

process of reverse transcriptase producing cDNA

template, primers, polymerase, nucleotides, and thermal cycler

wat 5 things does PCR require

denature DNA, anneal primers, extend primers (doubles the number of DNA molecules)

one cycle of PCR amplification

disease causing mutations or mutations to alter protein activity

what types of mutations can be introduced