TBL 3 DNA repair

Question 1

How is methylguane repaired and what causes it?

and what kind of DNA repair is it? and what enzyme repairs

Answer 1

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

Question 2

Direct DNA repair

Answer 2

Photoactivation in bacteria

Direct repair of alkylated bases

Direct repair DNA nick

Question 3

example of base repair mechanism

Answer 3

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

Question 4

example of Nucleotide excision repair

Answer 4

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

Question 5

Xeroderma pigmentosum

Answer 5

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

Question 6

mismatch excision repair

Answer 6

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

Question 7

what techniques are used for isolation?

Answer 7

Restriction endonuclease

Reverse transcriptase

Question 7

What DNA techniques are used for identification?

Answer 7

Probes
Gel electrophoresis
Southern blotting
Northern blotting
DNA sequencing
Next-generation sequencing

Question 8

techniques used for amplification of DNA

Answer 8

cloning, PCR, cDNA library

Question 9

How does Restriction Endonuclease work? and what is its use?

Answer 9

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.

Question 10

how does reverse transcriptase work?

Answer 10

uses mRNA to systhesize DNA, only has exons and not promoter or enhacors

Question 11

Gel electrophoresis

Answer 11

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.

Question 12

probes

Answer 12

hybridizes with DNA or RNA

Question 13

Southern vs Northern vs western Blotting

Answer 13

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.

Question 14

once synthesis of DNA sequencing is done? whats the next steps

Answer 14

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

Question 15

next generation sequencing steps

Answer 15

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

Question 15

DNA Sequencing uses

Answer 15

Involves the use of dideoxynucleotide (dNTPS, stops ddNTP)

• Determining mutation, insertion, deletion
• Mitochondrial profiling: maternal lineage
• Identifying genetic disorders
• Whole genome sequencing

Question 16

thalessemia what kind of DNA technology could be used?

Answer 16

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

Question 17

Cloning steps

Answer 17

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.

Question 18

PCR reqirements

application for PCR

Answer 18

DS DNA
taq polymerase
Mg2+
dNTPs
primers at 3’ of strand

used as allele specific probes

Question 19

qPCR or TR(reverse transcriptase)-PCR for disease diagnosis and example of disease

Answer 19

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

Question 20

Restriction Fragment Length Polymorphism (RFLP)

which disease can be diagnosed by this method?

Answer 20

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

Question 21

Allele-specific oligonucleotide (ASO) probes

and whats an example of a disease that can be diagnosed?

Answer 21

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

Question 22

what aa acid is changed in sickle cell?

Answer 22

GLU–> VAL

Question 23

Therapeutic proteins

Answer 23

insulin and growth hormones
- Plasmid vectors are engineered to
contain genes for insulin A-chain
and insulin B-chain.
- Engineered vectors are introduced
into Escherichia coli separately to
produce A chain and B chain of
insulin separately.
- Individual chains of insulin [A chain
and B chain] are purified, and then
linked together in a test tube.

Question 24

DNA vaccines

Answer 24

Hepatitis B vaccine (HBV)

not yet succesful in humans

Question 25

examples Gene Therapy

Answer 25

gene transfer, gene editing, silencing

Question 26

Gene transfer example and how it works

Answer 26

Gene trasfer can be done ex vivo (takes patient cell and introduced new gene via viral vector then returned ) or in vivo which the cells are directly transduced

ex: immunodeficiency disease caused by adenosine deaminase deficiency, impairs the development and function of immune cells

Question 27

Gene editing

Answer 27

CRISPR-CAS 9
for sickle cell patients

December 2023, the FDA approved Casgevy, the
first CRISPR-Cas9-utilizing gene therapy for the treatment of sickle
cell disease in patients 12 years of age and older with recurrent
vaso-occlusive crises.

Question 28

Gene Silencing (using small interfering RNA, siRNA):

Gene silencer drug

Answer 28

Inclisiran (Leqvio) - FDA-approved in 2021 to treat
heterozygous familial hypercholesterolemia or clinical
atherosclerotic cardiovascular disease as an add-on
therapy

Question 29

how does inclisiran (leqvio) work?

what the gene that is silenced?

Answer 29

“Silences” the gene (PCSK9) that codes
for PCSK9 (proprotein convertase
subtilisin/kexin type 9)

In the hepatocyte, inclisiran binds to an RNA-induced silencing complex  that
binds to PCSK9 mRNA  leading to
PCSK9 mRNA degradation  and
reduced production of PCSK9 protein.

decreases cholesterol levels

Question 30

western blot

Answer 30

Method:
1. Take a patient sample and separate proteins by gel electrophoresis
2. Add a primary antibody that is diagnostic for a disease
3. Wash and stain with the secondary antibody
4. Can be quantitative so the amount of substrate is determined