Recombinant DNA Flashcards
name the 4 important achievements leading to modern molecular biology
- Restriction endonucleases(enzymes)
- Cloning of DNA
- Creation of synthetic probes
- PCR
what do restriction endonucleases do?
- cleave very specific DNA sequences
- usually recognize sequences that are short (4-8 base pairs)
what are the short DNA sequences recognized by restriction enzymes called?
palindromes (also called restriction sequences)
how would you identify a palindrome?
-it is read the exact same (identical, not base pairs) from 5’ to 3’ direction on both strands
what are the two types of ends that can be formed when a restriction enzyme cleaves a DNA sequence?
- sticky ends
- blunt ends
which end is preferable for ligation? why?
sticky end!
-helps orient the DNA you want to ligate
how are restriction enzymes named?
-for the organism they were derived from
what is a restriction site?
- DNA sequence that can be cleaved by a restriction enzyme
- same thing as a palidrome
restriction enzymes that recognize LARGER sequences cut more or less frequently than the ones that recognize smaller sequences
-enzymes that recognize larger sequences cut less frequently
in general, restriction enzymes are…
tools that cut, paste, and analyze DNA
why is it important to know if the restriction enzyme recognizes larger or smaller sequences?
-if you are preparing DNA for a gel, you need to know how many fragments you are going to end up with.
Fragments of DNA can be “pasted” together to make a hybrid molecule known as…
-recombinant DNA
what enzyme creates the phosphodiester bonds that connect the sequences of DNA in the recombinant DNA?
DNA ligase
what are the two ways you can get recombinant DNA?
- generate it from PCR
2. get it straight from an organism
in the simplest sense, DNA cloning involves inserting a _________ into a ________
-inserting a restriction fragment (recombinant DNA) into a cloning vector
when is DNA considered “cloned”
as soon as it is inserted into the vector
what is a vector?
- molecules of DNA that can accept fragments of foreign DNA
- usually circular
what are the 3 things a vector must have?
- must be capable of replication in the cell (organism)
- must have at least one restriction site for foreign DNA
- must carry at least one gene for selection
what is the most common type of gene for selection used in a vector?
antibiotic resistance
why would you use two different restriction enzymes to cut the vector?
-to help the orientation of the inserted DNA
what is the most common vector? second most common?
- prokaryotic plasmids
2. yeast plasmids
what are the two different DNA libraries?
- Genomic DNA libraries
2. cDNA libraries
the collection of Genomimc DNA libraries contains what?
-all sequences in the genome, including coding regions, introns, promoters, etc.
how is the genomimc DNA library made?
-ENTIRE genome is chopped up by restriction enzymes, cloned into vectors, and used to transform bacteria
what is cDNA library also known as?
-complementary DNA library
how is cDNA generated?
- using isolated mRNA from a particular cell or tissue type
- mRNA is reverse transcribed and the second strand is synthesized
- it is then ligated into a vector, used to transform bacteria, and 1000s of clones are collected
what does cDNA library contain?
-sequences representing all mRNAs present in the cell or tissue type at the time the mRNA was collected
how much of the genome is represented with genomic DNA libraries? cDNA libraries?
genomic-representative of the entire genome
cDNA- only what was collected/present at that time
DNA from a cDNA library can be cloned into an expression vector for production of…
proteins!
what does the bacterial expression vector need to transform expression bacteria strains?
-promoter, shine-dalgarno sequence, and cDNA
why would you chose to use a yeast vector instead of a bacterial vector?
-when you want to express a protein that is integrated into the membrane
what is DNA sequencing used for?
to determine the exact sequence of a clones or PCR amplified stretch of DNA
what is used in DNA sequencing to stop DNA elongation? why?
- DIdeoxyribonucleotides
- ddCTP, ddATP, ddGTP, ddTTP
- they lack a free OH group, so elongation cannot continue
what are probes used for in general?
used to identify DNA fragments
what is a probe?
-single standed DNA molecule, labeled using radioactivity (or any label) that can be hybridized with a single stranded DNA molecule that is complementary
what is hybridization?
- target DNA is made ssDNA
- it is immobilized so it cannot reanneal
- then bathe it in probe
- probe binds to target
how is the target DNA immoblized?
- nitrocellulose membrane has charges associated with it that bind to the DNA
- it is hydrophobic in nature
what are small probes?
COMPLETELY CHEMICALLY synthesized oligonucleotides
-2-30 bp
what do small probes do?
- they are VERY specific
- can identify a single base mutation in a sequence
what are large probes?
-made by one of several molecular BIOLOGY techniques (reverse transcriptase, PCR)
what do large probes do?
- LESS specific
- can be used to identify similar genes in different organisms OR the same gene in different individuals
small probes are more________ and large probes are more_______
small probes are more diagnostic and large probes are more comparative
Southern blotting is the analysis of_______
DNA
Northern blotting is the analysis of_______
RNA
Western blotting is the analysis of______
Protein
what kind of sequences does Northern blotting detect?
-only EXPRESSED sequences
which two blotting techniques are quantitative and qualitative?
- Northern: measures what genes are expressed and how much they are expressed
- Western: measures what proteins are expressed and how much they are expressed
in Western blotting, the probe is a ______ and sees the protein as a _______
- the probe is an antibody
- the protein is an antigen
what is a polymorphism?
-genetic variation in non-coding region with no disease associated with them
what happens when there are genetic variations in intervening sequences?
- not much
- they are generally not harmful since they have no consequence on the expression of genes or proteins
why is the intervening sequence region hypervariable?
-there is no selective pressure for genetic changes in this region because they do not impact the expression of genes or proteins
what is a mutation?
genetic variations that actually cause disease
what are the two things that one or the other needs to happen in order to indicate a RFLP?
-the genetic change needs to create or destroy a restriction site
ORRRRR
-the genetic change needs to have more or less of a repeated sequence
what does RFLP stand for?
Restriction Fragment Length Polymorphism
what accounts for 90% of genetic variation?
single nucleotide polymorphisms (SNPs)
when would a single nucleotide polymorphisms (SNPs) cause a RFLP and when is it just a SNP?
- when it creates or abolishes a restriction site, it will create a RFLP
- when it doesnt, it is just a genetic varation
is an RFLP from a SNP usually created by a disease causing mutation?
- it can be
- more often its a harmless change that results in a different restriction pattern
with SNPs, is the new fragment longer or shorter?
- it depends!!
- it can create OR abolish a restriction site
- also depends on the original size of the fragment
how are RFLPs created with SNPs?
-creating or abolishing a restriction site
how are RFLPs created with VNTRs?
-more or less of a tandem repeat
what is the size of the RFLP dependent on when created by a SNP?
-depends on different restriction cutting
what is the size of the RFLP dependent on when created by VNTRs?
-depends on the number of repeats
what are RFLPs created by SNPs used for?
- used to mark genes
- disease markers
what are RFLPs created by VNTRs used for?
- used as molecular fingerprints
- used in forensics and paternity tests to identify individuals
how does DNA cloning amplify DNA?
-amplify fragments by inserting them into a vector and the HOST REPLICATES the vector
how does PCR amplify DNA?
- amplification all done completely in a tube
- amplify DNA without having to clone it
what do we need to create a primer for PCR?
-we need to know the sequence of two small flanking regions of the sequence we want to amplify
what are primers (used in PCR)
-nucleotides that are complementary to the flanking region of the target DNA
what enzyme do the primers for PCR act as a primer for?
DNA polymerase
what is used to denature the DNA for PCR?
Heat
what are the three steps of PCR that are repeated until the chain is the length you want?
- denature
- anneal
- extend
what are the general temperatures for denaturing, annealing, and extending?
- denaturing: 95 C
- annealing: 55 C
- Extension: 72 C
what kind of enzymes are used for PCR
heat stable Taq DNA polymerases
what are the two major advantages of PCR?
- high sensitivity: only need trace amounts of DNA to do it
- speed: only takes a few hours
- PCR gives really clean DNA
what are the two methods used for analysis of gene expression?
- northern blot
- microarrays
what is a microarray and what is used for?
- contain thousands of immobilized sequences (probes) on glass slides
- used to compare global gene expression changes in different cells types
what are the three ways to analyze proteins?
- proteomics
- enzyme-linked immunosorbent assays
- western blot
what is a major fault in ELISAs?
- they can produce false positives
- if negative, you can be confident in your results
- if positive, you should re check with western blotting
between western blotting and ELISAs, which one is most sensitive and which one is more specific?
- ELISA is more sensitive
- western blotting is more specific
what techniques are quantitative as well as qualitative?
- Northern blotting
- Western blotting
- Microarray
- ELISA
- Protenomics
Sickle cell is a mutation in what gene, and does what?
- mutation in B-globin gene
- eliminates a restriction site
sickle cell is an exception, but usually an RFLP used for diagnosis is linked to the disease, but not __________-
-no the actual disease causing mutation
what are the three ways to test for sickle cell?
- southern blotting
- PCR
- ASO probes
what are ASO probes?
- allele specific oligonucleotide
- they recognize allele specific differences
- hella specific
why is ASO awesome?
- specific and reliable
- very fast
- non-invasive
- inexpensive
