BIO282 Exam Already Passed
Bacteriophages - =Viruses that infect bacterial cells and once phage DNA enters the cell, usually
follows one of 2 life cycles:
1) Lysogenic Cycle where DNA enters cell and is inserted into bacterial chromosome and
remains inactive. Phages that reproduce this way are template phages and integrated phage is
prophage
2) Lytic Cycle where phage DNA replicates and reproduces more phage particles, released by
cell lysis. Prophage can change into this cycle. These are virulent phages as they kill host and
produce CPE plaques on lawn of bacteria
RNA Viruses - =Can either be ss or ds with ss being either negative requiring complementary
strands or positive or ambiguous-sense
Capable of integrating into the host genome such as retroviruses with integrated virus being
provirus
Prokaryotes vs. Eukaryotes - =Prokaryotes with bacterial genomes made of circular ds DNA that
is highly folded into a series of twisted loops
Eukaryotes with genomic DNA in the form of chromosomes with the number of chromosomes
different in every eukaryotes
Eukaryotic DNA Packaging - =DNA is organised with proteins name histones to create
chromatin. Histones are made of mostly basic amino acids which helps neutralise the negative
charge in DNA.
Chromatin wraps around histones (H1 - link) to create nucleosomes (H2A, H2B, H3, H4 used)
and then supercoiled in chromosomes.
Topoisomerase causes winding and unwinding during transcription and replication with either
type 1 (cut one strand) or type 2 (cut both strands).
Histones can be modified at amino-terminal tails
Heterochromatin is highly condensed and not transcribed
Euchromatin is less tightly coiled and mostly active genes
,DNA Structure - =Made of sugar, phosphate and base which can either be:
1) Purine (2 - carbon rings) either Adenine or Guanine
2) Pyrimidine (1 - carbon ring) either Cytosine, Thymine or Uracil
Nucleosides are the sugar and base while nucleotides are the nucleoside and phosphate
Type of DNA Sequence - =1) Unique sequence which only presents only once or only a few
times
2) Moderately repetitive sequence, 150-300bp repeated thousands of times, can have inter spread
repeats either being SINEs (short < 500bp) or LINEs (long > 5kb)
3) Highly repetitive sequence which are 10bp or less and repeated up to millions of times
Special RNA Structures - =Hairpin structure where nucleotides are inverted complements
Stem when the complementary strands are contiguous
Numerous structures can occur on the same RNA molecule
Tm Values - =Nucleic acids can be measured quantitatively using a spectrophotometer
Tm (meting temp.) is defined as the temperature at which half of ds DNA molecule are
denatured into single strands
Strands with more GC pairs have higher Tm due to GC pairs being held together by 3 hydrogen
bonds
DNA Cloning - =Used to produce a group of identical cells or organisms to either study a gene,
modify a gene or create transgenic organisms with genetic engineering
Done by:
1) Construction of recombinant DNA
2) Isolation and identification of desired recombinant
Vectors - =Autonomously replicating DNA molecules with unique restriction sites, marker that
distinguishes vector and selectable marker distinguishes between recombinant and non-
recombinant
,Plasmids are good being circular, super-coiled DNA either being relaxed (uncontrolled
replication) or stringent with regulated replication
Incompatibility states that plasmids can not co-exist in same cell
Plasmids can be either narrow (specific species) or broad (wide range of species)
Episomes can integrate into bacterial chromosomes
Conjugative plasmids mediate its own transfer to a new strain
Cryptic plasmids have no known function
Restriction Enzyme - =Restriction enzyme cut DNA at specific recognition sequences and leave
sticky ends. They are endonuclease with four types, Type 2 restriction endonuclease are used and
usually cut at palindromes
DNA Ligase - =Breaks in the sugar backbone to be repaired, catalyses reaction between 5'
phosphate of one strand and 3' hydroxyl of the other
Construction of Recombinant DNA - =1) Vector and foreign DNA are cleaved by some
restriction enzyme generating fragments
2) Mix together cut vector and insert DNA and ligate resulting in a mixture containing:
- Unligated vector
- Recirculaised vector
- Recombinant with underside DNA insert
- Ligated foreign DNA
- Desired recombinant DNA (wanted)
Transformation - =Used in isolation of desired recombinant after ligation by transferring mixture
into bacterial cells by:
- E.coli cells are made receptive by chemical treatments using competent cells
- DNA molecules get transferred inside of cells with one plasmid in one cell
- This creates a very large proportion of non-transferred cells and a small number of transferred
, Isolation and Identification of Desired Recombinant - =Transformants selected by vector having
antibiotic resistance when put on antibiotic media plate
Vector and recombinant plasmids distinguished by either:
1) Increased formation of recombinant plasmid be either using 2 different enzymes to digest
vector and insert and only way to re-circularise will be if it has both and treat the digested vector
with phosphatase creating linear DNA
2) Loss or gain of a function using restriction enzymes where it can disrupt lacZ gene responsible
for B-galactosidase which breaks down X-gal on media plate. Thus, insertion plasmids will
remain colourless while non-inserted plasmids will turn blue
DNA Modifying Enzymes - =Alkaline Phosphatase, removes phosphates from the ends of DNA
chain, useful in DNA cloning as ligase needs phosphate
Polynucleotide kinase, transfers gamma phosphate of ATP to 5' end of DNA, used to prepare for
ligation or labelling with gamma 32P ATP
Terminal deoxy-nucelotidyl transferase, adds nucleotides at the 3' ends of DNA and is template
independent DNA Polymerase. Used by adding homopolymeric tails in cloning or label single
nucleotides to 3' end to prepare probes
Klenom Fragment, a fragment of E.coli DNA Polymerase that has 3'-5' exonuclease and 5'-3'
polymerase, used in labelling or making blunt ends for cloning purpose
Nucleases - =Cut nucleic acid by breaking phosphdiester bonds, two categories:
1) Exonuclease which remove nucleotides one by one from the ends of DNA, number depends
on the incubation period. Can generate single stranded DNA and nested deletions
2) Endonuclease which break internal bonds within DNA e.g. Single Strand Specific Enzyme
(S1 Nuclease)
Non specific enzyme or restriction enzyme
DNA Replication - =The synthesis of DNA for:
- Mutations generation
- All organsims to grow
- Allows tissue to regenerate
- Development of diseases such as cancer
Bacteriophages - =Viruses that infect bacterial cells and once phage DNA enters the cell, usually
follows one of 2 life cycles:
1) Lysogenic Cycle where DNA enters cell and is inserted into bacterial chromosome and
remains inactive. Phages that reproduce this way are template phages and integrated phage is
prophage
2) Lytic Cycle where phage DNA replicates and reproduces more phage particles, released by
cell lysis. Prophage can change into this cycle. These are virulent phages as they kill host and
produce CPE plaques on lawn of bacteria
RNA Viruses - =Can either be ss or ds with ss being either negative requiring complementary
strands or positive or ambiguous-sense
Capable of integrating into the host genome such as retroviruses with integrated virus being
provirus
Prokaryotes vs. Eukaryotes - =Prokaryotes with bacterial genomes made of circular ds DNA that
is highly folded into a series of twisted loops
Eukaryotes with genomic DNA in the form of chromosomes with the number of chromosomes
different in every eukaryotes
Eukaryotic DNA Packaging - =DNA is organised with proteins name histones to create
chromatin. Histones are made of mostly basic amino acids which helps neutralise the negative
charge in DNA.
Chromatin wraps around histones (H1 - link) to create nucleosomes (H2A, H2B, H3, H4 used)
and then supercoiled in chromosomes.
Topoisomerase causes winding and unwinding during transcription and replication with either
type 1 (cut one strand) or type 2 (cut both strands).
Histones can be modified at amino-terminal tails
Heterochromatin is highly condensed and not transcribed
Euchromatin is less tightly coiled and mostly active genes
,DNA Structure - =Made of sugar, phosphate and base which can either be:
1) Purine (2 - carbon rings) either Adenine or Guanine
2) Pyrimidine (1 - carbon ring) either Cytosine, Thymine or Uracil
Nucleosides are the sugar and base while nucleotides are the nucleoside and phosphate
Type of DNA Sequence - =1) Unique sequence which only presents only once or only a few
times
2) Moderately repetitive sequence, 150-300bp repeated thousands of times, can have inter spread
repeats either being SINEs (short < 500bp) or LINEs (long > 5kb)
3) Highly repetitive sequence which are 10bp or less and repeated up to millions of times
Special RNA Structures - =Hairpin structure where nucleotides are inverted complements
Stem when the complementary strands are contiguous
Numerous structures can occur on the same RNA molecule
Tm Values - =Nucleic acids can be measured quantitatively using a spectrophotometer
Tm (meting temp.) is defined as the temperature at which half of ds DNA molecule are
denatured into single strands
Strands with more GC pairs have higher Tm due to GC pairs being held together by 3 hydrogen
bonds
DNA Cloning - =Used to produce a group of identical cells or organisms to either study a gene,
modify a gene or create transgenic organisms with genetic engineering
Done by:
1) Construction of recombinant DNA
2) Isolation and identification of desired recombinant
Vectors - =Autonomously replicating DNA molecules with unique restriction sites, marker that
distinguishes vector and selectable marker distinguishes between recombinant and non-
recombinant
,Plasmids are good being circular, super-coiled DNA either being relaxed (uncontrolled
replication) or stringent with regulated replication
Incompatibility states that plasmids can not co-exist in same cell
Plasmids can be either narrow (specific species) or broad (wide range of species)
Episomes can integrate into bacterial chromosomes
Conjugative plasmids mediate its own transfer to a new strain
Cryptic plasmids have no known function
Restriction Enzyme - =Restriction enzyme cut DNA at specific recognition sequences and leave
sticky ends. They are endonuclease with four types, Type 2 restriction endonuclease are used and
usually cut at palindromes
DNA Ligase - =Breaks in the sugar backbone to be repaired, catalyses reaction between 5'
phosphate of one strand and 3' hydroxyl of the other
Construction of Recombinant DNA - =1) Vector and foreign DNA are cleaved by some
restriction enzyme generating fragments
2) Mix together cut vector and insert DNA and ligate resulting in a mixture containing:
- Unligated vector
- Recirculaised vector
- Recombinant with underside DNA insert
- Ligated foreign DNA
- Desired recombinant DNA (wanted)
Transformation - =Used in isolation of desired recombinant after ligation by transferring mixture
into bacterial cells by:
- E.coli cells are made receptive by chemical treatments using competent cells
- DNA molecules get transferred inside of cells with one plasmid in one cell
- This creates a very large proportion of non-transferred cells and a small number of transferred
, Isolation and Identification of Desired Recombinant - =Transformants selected by vector having
antibiotic resistance when put on antibiotic media plate
Vector and recombinant plasmids distinguished by either:
1) Increased formation of recombinant plasmid be either using 2 different enzymes to digest
vector and insert and only way to re-circularise will be if it has both and treat the digested vector
with phosphatase creating linear DNA
2) Loss or gain of a function using restriction enzymes where it can disrupt lacZ gene responsible
for B-galactosidase which breaks down X-gal on media plate. Thus, insertion plasmids will
remain colourless while non-inserted plasmids will turn blue
DNA Modifying Enzymes - =Alkaline Phosphatase, removes phosphates from the ends of DNA
chain, useful in DNA cloning as ligase needs phosphate
Polynucleotide kinase, transfers gamma phosphate of ATP to 5' end of DNA, used to prepare for
ligation or labelling with gamma 32P ATP
Terminal deoxy-nucelotidyl transferase, adds nucleotides at the 3' ends of DNA and is template
independent DNA Polymerase. Used by adding homopolymeric tails in cloning or label single
nucleotides to 3' end to prepare probes
Klenom Fragment, a fragment of E.coli DNA Polymerase that has 3'-5' exonuclease and 5'-3'
polymerase, used in labelling or making blunt ends for cloning purpose
Nucleases - =Cut nucleic acid by breaking phosphdiester bonds, two categories:
1) Exonuclease which remove nucleotides one by one from the ends of DNA, number depends
on the incubation period. Can generate single stranded DNA and nested deletions
2) Endonuclease which break internal bonds within DNA e.g. Single Strand Specific Enzyme
(S1 Nuclease)
Non specific enzyme or restriction enzyme
DNA Replication - =The synthesis of DNA for:
- Mutations generation
- All organsims to grow
- Allows tissue to regenerate
- Development of diseases such as cancer
