TBL 3 DNA repair Flashcards
How is methylguane repaired and what causes it?
and what kind of DNA repair is it? and what enzyme repairs
Happens when too much S-adenomethinine is produced by the cell and when dimethyl sulfate is used in the environment.
It add a methyl group to the guanine.
methyl transferase will fix the issue if not the gene will be silenced
Direct DNA repair
Direct DNA repair
Photoactivation in bacteria
Direct repair of alkylated bases
Direct repair DNA nick
example of base repair mechanism
When 8-oxoguanine is inserted instead of a Tdue to oxidative species or toxic environmental factors like steel
the body will repair with the enzyme Human 8-Oxoguanine glycosylase recognizing 8- oxoguanine (HOGG1). The 8-oxoguanine will be removed producing a AP then AP endonuclease willcleave it and DNA pol will add the correct base, ligase will seal
example of Nucleotide excision repair
Benzo (a)pyrene, disrupts bases of guanine
when more than nucleotide are damaged, enzyme complex will recognize mistake and endonuclease comes and cuts, DNA helicase will cut it out, DNA pol will add bases on the 3’. DNA ligase seals
Xeroderma pigmentosum
The body has mutations in the enzymes that code for nucleotide excision repair mechanism (xeroderma pigmentosa)
patients develop an accumulation of mutations
example of what happens when nucleotide excision repair mechanism is non-functional
mismatch excision repair
when the DNA pol makes a mistake and adds a different base. mismatch repair enzymes- MUT L,S,H) find the bad base new strand because it is not methylated. If mutation is missed and mitosis happens strands will separate and mutation is permanent.
mutations in repair enzymes lead to Hereditary non-polyposis colorectal cancer or Lynch Syndrome
loss of function mutation
what techniques are used for isolation?
Restriction endonuclease
Reverse transcriptase
What DNA techniques are used for identification?
Probes
Gel electrophoresis
Southern blotting
Northern blotting
DNA sequencing
Next-generation sequencing
techniques used for amplification of DNA
cloning, PCR, cDNA library
How does Restriction Endonuclease work? and what is its use?
Enzymes that cut DNA at a specific site, when read from 5’ to 3’ the sequences are the same.
Restriction fragments of DNA can be used to identify variations in base
sequence in a gene. different cuts, different genes
They also can be used to synthesize recombinant
DNA (or chimeric DNA) - DNA from different sources that have been
recombined in vitro. Two unrelated DNA fragments are cut using the
same restriction enzyme to produce complementary sticky ends. The
sticky ends base-pair with each other and are joined by DNA ligase.
how does reverse transcriptase work?
uses mRNA to systhesize DNA, only has exons and not promoter or enhacors
Gel electrophoresis
separates based on size. smaller molecules move toward the bottom after electric field has been applied
Steps:
1) DNA samples are placed into depressions (“wells”) at
one end of a gel (typically agarose gel), and an electrical
field is applied.
2) The DNA migrates toward the positive electrode at a
rate that depends on the size of the DNA molecules.
3) As the gel acts as a sieve, shorter molecules migrate
more rapidly than longer molecules.
4) The gel is removed from the apparatus. The bands are
visualized using staining and visualization techniques.
probes
hybridizes with DNA or RNA
Southern vs Northern vs western Blotting
Southern blots:
- DNA molecules are separated by electrophoresis, denatured, transferred
to nitrocellulose paper (by “blotting”), and hybridized with a DNA probe
DNA sequences are visualized.
Northern blots:
- RNA is electrophoresed and treated similarly except that alkali is not
used (first, because alkali hydrolyzes RNA, and second, because RNA is
already single-stranded) RNA sequences are visualized.
Western blots:
- One of the early-generation tests for diagnosis of HIV.
- Proteins are electrophoresed, transferred to nitrocellulose, and probed
with a specific antibody Specific proteins are visualized.
once synthesis of DNA sequencing is done? whats the next steps
the pieces of DNA are subject to electrophoresis and are read from the bottom up.
ddNTPs are radioactive to indicate where it has ended
then laser
next generation sequencing steps
faster than traditional DNA sequencing
DNA is fragmented and an adaptor is added to it that wil bind to a complementary sequence on flow cell.
PCR then it forms clusters of that DNA sequence then one nucleotide issynthesized at a time with fluorophore
laser
data analyzed
DNA Sequencing uses
Involves the use of dideoxynucleotide (dNTPS, stops ddNTP)
- Determining mutation, insertion, deletion
- Mitochondrial profiling: maternal lineage
- Identifying genetic disorders
- Whole genome sequencing
thalessemia what kind of DNA technology could be used?
lower than normal hemogloblin levels
alpha (poorly formed tetramers
or beta (different types of mutations)
autosomal recessive
Next generation sequence, can tell you all subclasses at once
Cloning steps
Steps:
1) Cleave the DNA of interest and plasmid vector containing
antibiotic resistance genes, with the same restriction enzyme.
2) Insert DNA of interest into the plasmid to create a chimeric
plasmid.
3) Insert plasmid into bacteria with antibiotic media.
4) Surviving bacteria must express protein of interest.
5) To obtain cloned DNA: isolate plasmids and cleave with
restriction endonuclease.
6) To obtain protein: grow under conditions that allow
expression of cloned DNA isolate protein.
PCR reqirements
application for PCR
DS DNA
taq polymerase
Mg2+
dNTPs
primers at 3’ of strand
used as allele specific probes
qPCR or TR(reverse transcriptase)-PCR for disease diagnosis and example of disease
used to determine gene expression
Prader Willi Syndrome expression of UBE3A mRNA gene expession
-one gene is mutated and the other is muthylated at promoter region so the gene is not expressed and no green color will show
Restriction Fragment Length Polymorphism (RFLP)
which disease can be diagnosed by this method?
used to detect restriction site mutation
people with point mutations in recognization sites will not be able to be cut by restriction sites and therefore the cut segment will be larger
mutation creating new sites will be cut short
Sickle cell disease cause by a mutation in beta hemoglobin gene and a defective b hemoglobin chain is produced. restriction site is missing in sickle cell
Allele-specific oligonucleotide (ASO) probes
and whats an example of a disease that can be diagnosed?
For any type of muation
DNA from patient is taken and if the probe hydridizes with patient DNA, then positive test for diease
if negative test, then no disease
sickle cell can be diagnosed this way also
what aa acid is changed in sickle cell?
GLU–> VAL
