Chapter 6.5 Flashcards
What is meant by recombinant DNA technology
-it describes how a DNA fragment from any source can be multiplied by gene cloning or polymerase chain reaction(PCR)
Is DNA to be cloned present in a small quantity? Is it a heterogenous mixture or a homogenous one?
- it is present in small quantities
- >it is a heterogenous mixture to start off with
What are vectors? Describe the process of transferring it into a host bacteria.
- they are bacterial or viral plasmids
- > that can be transferred into another bacterium(the host)
- > bacteria are then grown in colonies and a colony containing the recombinant vector is isolated
- > note that the recombinant vector also contains a gene for antibiotic resistance
-note vectors also contain palandromic sequences that can be recognized by restriction enzymes
What are restriction enzymes? What kind of strands do they recognize?
- they are enzymes that recognize specific double stranded DNA sequences
- > these sequences are palindromic(meaning that five prime to 3 prime sequence is identical to three prime to five prime strand)
Where are restriction enzymes isolated from?
- they are isolated from bacteria
- >bacteria is their natural source
What are DNA libraries? How are they made
- large collection of known DNA sequences
- > it could equal to the genome of an organism
- they are made because DNA fragments are often digested randomly
- > are cloned into vectors and utilized for further studies
What are genomic libraries
- contain large fragments of DNA
- > include both exon and intron(non coding regions) of DNA
-may contain multiple vectors
What are cDNA libraries? How are they made?
- libraries constructed by reverse-transcribing processed mRNA
- > therefore, cDNA lacks noncoding regions such as introns
-note cDna libraries use reverse transcriptase enzymes, while genomic libraries use restriction endonuclease
What is meant by the process of hybridization?
- joining of complementary base pair sequences
- this can be DNA-DNA recognition or DNA-RNA recognition
-uses two single stranded sequences
How does PCR work? Discuss primers
- they got primers complementary to the DNA
- > primers have high GC content and therefore are stable
- during PCR
- > DNA is denatured
- > replicated
- > and then cooled to allow reannealing of the daughter strands with the parent strands
- > this process is repeated several times, doubling the amount of DNA with each cycle
-the DNA polymerase used for PCR is Thermus aquaticus
What kind of gel is used for gel electrophoresis of DNA?
-agarose gel is used
What is southern blotting? Describe the process
-it is used to detect the presence and quantity of various DNA strands in a sample
- DNA is cut by restriction enzymes
- > then separated by gel electrophoresis
- > DNA is then transferred to a membrane
- > membrane is then probed with many copies of single-stranded DNA sequence
- probes bind to complementary sequences and form double stranded DNA
- > probes are labelled with radioisotopes or indicator proteins
- > which can be used to indicate the presence of a desired sequence
What materials or products are needed for DNA sequencing?
-you need template DNA, primers, an appropriate DNA polymerase, all four doexyribronucleotide triphosphates, and dideoxyribonucleotides in low concentrations
What is so special about dideoxyribonucleotides?
- they contain a hydrogen at carbon 3 rather than a hydroxyl group
- > so the polymerase can no longer add to this chain
- eventually, you end up with many fragments
- > each one which terminates with one of the modified dideoxyribonucleotides
- > these fragments are then seperated by size using gel electrophoresis
- > the last base for each fragment is read
