Mutations and recombinant DNA Flashcards
Triplet expansion
600 repeats of CCG over and over- this is not a large scale chromosomal abberation.
- Fragile X syndrome
- Huntingdon’s Disease
- Myotonic dystrophy
in general DNA has high fidelity because of:
editing function
spacing in base pairing
pairing rules
DNA repair mechanisms
spontaneous mispairings of bases
An A accdientally goes with a C or G instead of a T
point mutations list
transition, transversion, missense, nonsense, neutral, silent, frameshift
point mutations: transition mutation
AT to GC- keeps the same type of nucleotid (eg G is still a purine)
transversion
CG -> GC (this actually changes the type of nucelotide)
missense mutation
change amino acids entirely ; AAA-> GAA goes from lysine to glutamic acid
nonsense mutation
changes an amino acid into a stop codon. this could happen via transversion of just one pair
neutral mutation
change from one AA to another with similar chemical properties (ie lysine to arginine, both basic)
silent mutation
same AA, diff configuration - NO BIGGIE!
frameshift mutation
change in reading frame due to addition/substraction of some bases- BIG DEAL!
polymorphism definition and examples
change in genotype but not phenotype.
ex: neutral, silent mutations
xeroderma pigmentosum
Affected individuals lack excision repair enzymes and fail to remove thymidine dimers
people who lack the enzyme to repair skin’s UV damage due to messed up pairings in bases. shows importance of enzymatic repair activity
recombinant DNA creation process
restriction enzymes cut DNA at specific sequences- isolate plasmids, stick in a piece of foreign DNA, put back into bacteria cell and let it replicate
whats the deal with palindromic restriction enzymes?
The recognition sequences are always palindromic
EcoRI for example is GAATTC- when you read complementary sequence its CTTAAG, (5’ to 3’) palindromic to its original sequence
Plasmid:
A small (1000 – 10,000 bp) circular dsDNA that is distinct from the normal chromosomal DNA
why are the recognition sequences for restriction enzymes palindromic?
Enzymes act as dimers. One recognizes top sequence , other part of enzyme recognizes that bottom guy, also GAATTC. Dimer- just means enzyme/protein that has two parts
what skill have bacteria evolved to protect their DNA from being chewed up?
methylate the ends of their DNA to protect from enzymes
recognize the same sequence as restriction enzyme, put a methyl groups on it.
when cutting DNA with a restriction enzyme you get sticky ends- what are those?
- get an overhang of either 5’ or 3’
Sometimes you have a piece of DNA that doesn’t have the right ends- now you have to insert short little sequences called ____ (6 bases long, contains recognition sequence) that have the right sequences
linkers
whats the flow of recombinant process
DNA -> Vector -> Host
options for DNA? options for vector? host?
DNA
- Genomic
- cDNA
Vector
- Plasmid
- Bacteriophage
Host Cell
- Bacterium
- Eukaryotic cells in culture
explain how the linkers help you in the process of cloning
cloning: DNA pieces goes into plasmid, host cell replicates and produces copies. sometimes the piece of DNA needs to be modified first.
You’d ligate BAMHI linkers onto the ends of the DNA piece, cut with BAMHI enzyme. Gives us sticky ends. Took a plasmid with BAM recognition site, cut it to get sticky ends, and THAT will be ligated- to get a recombinant DNA
- Purpose of cloning:
to get a sequence of DNA that is identical in multiple copies
how do you do a test to select where your plasmids with inserts are?
Put beta galactosidase ‘lac Z gene’ into plasmid. (Borrowed it from a lac operon.) Stuck in in with a promoter. Put polylinker in such a way so that lacZ gene still works. allow functional β- galactosidase to be made. Note: lac Z turns surrounding environment blue if it is being transcribed/translated properly. If not, stays clear. Use this enzyme so that you can use blue/white test to determine whether a sequence has been inserted in here. The cells that got plasmids inserted into them now have non functional lac - medium will stay clear- vs ones without lac turn blue.
- Take bacterial samples, test them on a plate that has ampicillin. All colonies without a plasmid will die. 2) And then take a sample of each of those, test on x-gal , see who turns blue and who is white. Want the white ones, have an insert.
- We’re left with only ones with plasmids with inserts!
cDNA- formation and uses
In genetics, complementary DNA (cDNA) is DNA synthesized from a single stranded RNA (e.g., messenger RNA (mRNA) or microRNA) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to clone eukaryotic genes in prokaryotes.
give some examples of vectors
a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed
= plasmid or bacteriophage
Cloning in bacteriophage (lambda phage)
Can insert DNA into the phage. It puts it into its capsid, and then infects bacteria, who then all become phage production factories. Make thousands of copies. Isolate sequence of interest.
Phage dna is ___ (linear/circular) . Bacteria dna is circular.
linear
library of genes
all bacteria containing all sizes of recombinant DNA OR all phages containing all sizes of recombinant DNA
Genomic DNA and cDNA – create libraries of genes. Put these into plasmids or phages.
Screen library by Hybridization
use radioactive probe to identify specific sequences
radioactive stuff only sticks to guys that have tubulin sequence. Detect this using x-ray films – called “autoradiography”
A black spot will appear- that colony is the one with the tubulin sequence
what are the black colonies here?
screening library by hybridization- Positive colonies are in black, negative colonies are that fuzzy grey. positive means it has the desired tubulin sequence- aka, its the colony containing the desired gene
in gene library screening, what does the probe do
the probe will bind to any clone containing sequences similar to those found on the probe. This binding step is called hybridization.
what exactly is the probe in genetic library screening?
A probe is normally a cloned piece of DNA that contains a portion of the sequence for which you are searching. You typically will make the probe radioactive and add it to a solution.
Screen library by looking for expression of a specific gene product:
- use special plasmid with a promoter sequence for transcription in bacteria (RNA polymerase needs to know where to sit down and start)
- put plasmid into bacteria, grow the colony, put samples of each colonies on filters – which of these colonies are expressing the protein of interest?? Will detect using antibodies- radioactive antibody. Antibody sticks only to the colonies that are making tubulin. Do the autoradiograph (product u get when u use x-ray film) and identify clone.
- Radioactive antibody probe sticks to PROTEIN
how do you use ampicillin to test for presence of plasmid after transformation?
Ampicillin sensitive : plasmid absent (died)
Ampicillin resistant colonies : plasmid present
how can you test for presence of a DNA insert in the plasmid
a. Blue/White test. A functional β-galactosidase gene will turn an indicator dye (X-gal) blue; a “white” colony (more accurately beige, like the medium) on the other hand, shows that an insert is present and has interrupted the β-gal sequence and inactivated it.
b. Second antibiotic test. E.g. Tetracycline resistance – if the cloning site is in the middle of the Tet gene then an insert will make the bacterial host sensitive to tetracycline (Tets). In this case the transformed bacterial cells would be Ampr and Tets.
c. Hybridization screening with a DNA probe. Positive hybridization signal (i.e. a spot on the X-ray film) identifies which, if any, of these colonies contains a sequence complimentary to your radioactive DNA probe.
d. Expression screening
–Requires your vector to have a promoter sequence adjacent to the insertion site so that transcription can occur (and maybe a termination sequence).
–If the inserted gene sequence is in the correct orientation and is transcribed and translated by the host cell it can then be detected by:
•Test for presence of protein product by using an antibody probe to confirm its presence.
DNA electrophoresis description and purpose
- Process of causing DNA molecules 2 move thru gel in response 2 electric field. Put the samples in the little notches in the gel.
- separates DNA by size (tiniest ones move farthest)
Southern Blotting
- Wanted to be able to use hybridization to see which fragment in electrophoresis was of interest
- A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
Northern blotting:
southern blotting but with RNA
Western blotting:
Southern blotting with proteins
Restriction mapping:
- Allows you to locate the relative positions of _______ in a DNA sample
restriction enzyme cut sites
polymerase chain rxn
a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
why do you use Taq in PCR?
PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called Taq polymerase,
T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-stable and is most active around 70°C
does Taq polymerase require a primer to begin PCR?
yes
the three main steps of PCR
The basic steps are:
Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.
Annealing (55-65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
T/F: PCR Only amplifies short pieces of DNA (300-1,000 nucleotides in length)
true
chain termination technique for DNA sequencing
-requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleosidetriphosphates (dNTPs), and modified di-deoxynucleotidetriphosphates (ddNTPs), the latter of which terminate DNA strand elongation.
- These chain-terminating nucleotides lack a 3’-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively or fluorescentlylabeled for detection in automated sequencing machines.
- The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase
- Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis.
- The DNA bands may then be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image.
Restriction Fragment Length Polymorphism (RFLP)
- Genetic testing to look for differences in genome sequence that people might have. Eg sickle cell anemia
- Comparing beta globin gene between individuals. Most of us have 2 copies of normal gene, sickle cell can have 2 recessive alleles. Difference between these two forms was a change in amino acid #6- single point mutation. That changes restriction enzyme recognition site (DdeI) who would normally cut it up. Somehow probe only detecs the DdeI at 201 base pairs and 175 base pairs- if u see that, normal arrangement of genes. If u get a piece 376 bases long u know u have sickle cell. If you were heterozygous, you’d get one piece 201 and 175, and one at 376 as well.
- Because the sequence changes In the sickle cell allele, distinguish sickle cell vs normal allele quickly. basis for genetic tests.
VNTR’s / STR’s
- Variable between diff people, but looks the same between families: Can be used to do maternity/paternity analysis
- Find sequences of a lot of variation. Region of genome with sequence repeated multiple times- some ppl have fewer repeats others have more. Think of these as alleles in that location.
- Get different bands based on where their genes repeat- if homologus chromes at 10, get a band at 10
Paternity testing using recombinant DNA technology
- DNA probe contains sequences for a specific VNTR region
- Take sample from baby’s mom and suspected father
- Use a probe- develop autoradiogram – see a banding pattern. Baby has an allele perfectly matching mother and has another band perfectly matching baby. This shows that this man is POSSIBLLY the father- we can’t say for sure. If he had no bands matching baby , could NOT have been the father. Its really only good at exclusion
DNA fingerprinting analysis for forensic analysis
- Take semen from crime scene and blood from suspect. See a whole banding pattern (13 diff loci) see if suspects blood matches the semen.
- 1/72 billion odds that these match aka has to be the same person
- basically you cut with several different restriction enzymes to get matches – if you can see multiple matches that’s evidence
how can we use PCR to solve a crime?
use PCR to amplify regions based on a single strand of hair- combine with forensic analysis to have a lot of DNA, information
Recombinant DNA in useful products: (eukaryotes)
insulin, growth hormone, clotting factors
gene therapy- how does it involve cloning?
Gene Therapy
e.g.
Recombinant mice that have received growth hormone gene
genetic modification- how is this related to recombinant DNA?
Enzymes for b-carotene synthesis are transferred into rice from daffodils
b-carotene, a precursor to vitamin A, is synthesized.
