Download Genetic Investigation Techniques

CLASS: 11-12
DATE: 9-22-2010
PROFESSOR: Pittler
I.
LECTURE TITLE
Scribe: Jordan Taylor
Proof: Lee Redditt
Page 1 of 4
Receiving a Too-Strong Division Signal [S29]
a. There is rearrangment of the genome in some way.
i. inversion, translocation, etc
II. Oncogenes: new functions from old [S30]
a. Proto-oncogene – normal gene
b. Gene is expressed in brain
c. Fusion – when the promoter of the brain protein binds with proto-oncogene to create inappropriate
response.
d. This gene is being expressed normally, but it’s being expressed in the wrong location (brain)
III. Tumor suppressor genes [S31]
a. Point mutations are rare, but they can occur
b. Two mutations of tumor suppressor genes are usually required to allow cell division (cancer), so with
only one mutation the gene is still able to suppress division
IV. Retinoblastoma [S32]
a. One eye – unilateral, Two eyes – bilateral; there’s also a trilateral – involves the pineal gland
b. Age of diagnosis – when did the patient first come in?
c. In developed countries we generally don’t see extreme cases of retinoblastoma in which the eye is
severely protruding; this is more common in third-world nations
d. Relatives – look at the genetic pedigree
e. How many individual tumors can be recognized per eye?
f. Unilateral cases are tougher to diagnose than bilateral cases because generally if one eye is normal,
it will compensate for the deficiency in the diseased eye, making the problem less noticeable. This
can’t occur in bilateral cases.
V.
Knudson’s two hit hypothesis [S33]
a. Retinoblastoma is the most frequent ocular tumor that is known.
b. Applies to many cancers, not just retinoblastoma
c. Sporadic cases – two somatic mutations must occur, one in each gene copy
d. Familial cases – one mutation is inherited and one mutation occurs somatically, this is more common
than sporadic cases.
e. The likelihood of one somatic mutation is much higher than two. This is why sporadic cases are
more rare.
VI. p53 coordinates cell cycle regulation [S34]
a. Li-Fraumeni (Li-F) syndrome – involves mutation of the p53 gene, which expresses protein important
to cell cycles. This inherited disease results in a variety of cancers at early ages.
VII. Environmental trigger (diagram) [S35]
a. items on the left are “triggers”, they affect the p53 gene to lead to different cancers
b. items on the right are targets of the cancer
VIII. BRCA1, a breast cancer susceptibility gene [S36]
a. Mutation of one copy of this gene (inherited) does not guarantee cancer, but it definitely increase
susceptibility
b. An inherited mutational copy plus a somatic mutation will lead to tumor, therefore, BRCA1 acts in a
recessive manner (follows the “two-hit hypothesis”)
IX. Complexities: Genetic Counseling of Breast Cancer (table) [S37]
a. “Be able to distinguish between somatic and familial cases”
b. What is incompletely penetrant? Just because you have the mutation/change doesn’t mean you have
the disease. BRCA gene is not causative, a mutation here only increases susceptibility
c. Example: One person (A) has mutated gene and disease. Another person (B) has mutated gene, but
no disease. The gene is incompletely penetrant. In person B, some other factor in the body (possibly
another gene) is compensating for the lack of function in the mutated gene. Therefore, no disease is
present in B.
d. Most classic types of genetic disorders are incompletely penetrant.
e. Incompletely penetrance is rarely found in lab mice. Why is it more prevalent in humans? Because,
human genetic backgrounds are much more diverse. Lab mice are generally bred in a controlled
fashion to produce large numbers among a closed population (very little change in the genes).
Humans have a greater variety of genetic backgrounds so this variety will influence the severity of the
effect of a particular mutation.
X.
Inheritance Risk of BRCA1 Mutation (table) [S38]
CLASS: 11-12
Scribe: Jordan Taylor
DATE: 9-22-2010
Proof: Lee Redditt
PROFESSOR: Pittler
LECTURE TITLE
Page 2 of 4
a. Genetic counseling is used to discuss risks and potential genetic abnormalities that could occur for a
particular couple based on their family history.
XI. Cancer Genes (table) [S39]
a. Don’t memorize this table. Just to show that there are numerous types of oncogenes, tumor
suppressor genes, etc.
XII. Multiple genes contribute to cancer progression [S40]
a. Cancer can occur from a change in one gene, or some cancers require mutations or altered function
of multiple different genes
b. All of the mutations seen on this slide can result in an astrocytoma (type of brain cancer)
XIII. Colon cancer results from genetic alterations [S41]
a. Colon cancer is an example that requires at least four different genes that contribute to uncontrolled
cell growth
b. Also an example of a susceptibility gene. An inheritance of a mutation in one of these genes will
increase the likelihood of receiving a second hit on another gene and so forth, resulting in cancer
XIV. Environment impacts cancer [S42]
a. Carcinogens (smoking)
b. Radiation (sunlight)
c. Diet (fatty foods)
d. All of these environmental factors can play a role in cancer progression
XV. Cruciferous vegetables can lower cancer risk [S43]
a. Burgers can produce heterocyclic aromatic amines, which can be metabolized into mutagens, which
can ultimately lead to an increase in colon cancer.
b. Cruciferous vegetables (brussel sprouts, broccoli) can counter this by producing glucosinolates
which activate xenobiotic metabolizing enzymes, which can metabolize these heterocyclic aromatic
amines into non-harmful non-mutagenic metabolites
XVI. Methods for evaluating environmental impacts [S44]
a. Know the differences between these different types of studies, but we will not go in depth.
XVII. Increase in death rates for certain cancers (table) [S45]
a. There are clear regional correlations to cancer.
b. There are a number of different factors that can contribute to the prevalence of cancer such as
chemical plants, asbestos exposure, factories, etc.
XVIII. Cancer treatments for breast cancer [S46]
a. The goal is to get rid of 100% of the cancerous cells. If you do not remove the cancer completely, it
can re-grow and metastasize.
b. We can destroy cancerous tissue by chemotherapy/radiation.
c. The aim is to destroy the most rapidly dividing cells. This is why treatment individuals experience
hair loss, fingernail loss, etc.
d. Treatment can use a drug and target it towards a particular phenotype (Estrogen receptor) or
genotype (Her-2/neu gene). The drug can inhibit these erroneous targets.
XIX. Most genetic technologies are based on four properties…[S9] (begin next presentation)
a. S1-S8 (skipped)
b. What are the 4 properties of DNA?
i. DNA can be cut at specific sites (sequence motifs) by restriction enzymes.
1. Restriction enzymes are part of bacterial immune systems, protecting their DNA.
ii. Different lengths of DNA can be size-separated by gel electrophoresis.
1. Gel with electric field imposed upon can separate pieces of DNA.
iii. Single strand of DNA will stick to its complement.
1. Denature DNA in to single strands, use a radioactive/fluorescent DNA piece to target a
particular sequence, they will anneal/hybridize together
iv. DNA can be copied by a polymerase enzyme (PCR)
c. Some of these properties apply to RNA as well.
XX. DNA can be cut at specific sites by an enzyme [S10 – S13]
a. The enzyme (ie: Sau3A) will target a particular recognition sequence (often palindromic)
b. It splits the DNA in a specific manner. They either stagger the cut: leave a 5’ or 3’ overhang, or
bluntly cut (no overhang).
c. These are “true enzymes” because they act on more than one site. They cut everywhere they
recognize a particular sequence.
CLASS: 11-12
Scribe: Jordan Taylor
DATE: 9-22-2010
Proof: Lee Redditt
PROFESSOR: Pittler
LECTURE TITLE
Page 3 of 4
d. Now we have two pieces of DNA that can be ligated back together by DNA ligase. Or DNA can be
ligated to other strands that were cut by the same enzyme or a similar enzyme.
XXI. DNA can be cut at specific sites by an enzyme [S14]
a. Notice the different types of cuts: 5’, 3’, blunt-end (no overhang)
XXII. Different lengths of DNA can be separated by gel electrophoresis [S15]
a. DNA is negatively charged and begins to move towards the positive when electric field is applied
b. We take the DNA after it’s been cut by restriction enzymes and use electrophoresis to separate the
strands
c. We will see a band at every point on the DNA strand that was cut by the restriction enzyme
d. Use a marker (a standard of known lengths) to determine the size of each cut DNA strand
XXIII. Different lengths of DNA can be separated by gel electrophoresis [S16]
a. Smaller pieces of DNA move faster through the gel (less hindrance)
XXIV. Different lengths of DNA can be separated by gel electrophoresis [S17]
a. In this case, the restriction enzyme doesn’t cut the recessive (mutant) DNA strand because the
recognition sequence has been altered
b. Therefore, we can use this method to determine if a particular gene is mutated or not, used to
genotype patients
XXV.
Different lengths of DNA can be separated by gel electrophoresis [S18]
a. HH = Homozygous normal, HD = heterozygous, DD = homozygous mutant
b. (red box) Only one strand size is found in the HH, only one strand of a different size is found in the
DD, and both strands are found in the HD.
XXVI. A single strand of DNA will stick to its complement [S19-S22]
a. At 95 degrees, the DNA will “melt” and strands will separate from one another. At 60 degrees, they
will re-anneal.
XXVII.
A single strand of DNA will stick to its complement [S23]
a. Putting the whole process together, we take a swab of a patient’s throat to gather a DNA sample. This
gives the whole genome, massive amounts of DNA. We need to isolate the particular gene sequence
that we’re interested in among the enormous supply of information.
i. Digest all DNA with restriction enzyme
1. Enzyme will cut at numerous places
ii. Separate on agarose gel
iii. Stain with Ethidium Bromide
1. Stains everything. Results in a smear because there’s so much DNA. We can’t distinguish
individual bands.
iv. Solid Support Transfer (Southern blotting)
v. Probe for particular sequence with labeled DNA (radioactive/fluorescence)
XXVIII. A single strand of DNA will stick to its complement [S24]
a. Southern blotting – sandwich the agarose gel with DNA in between a membrane (bottom) and wet
paper (top). Then place dry paper underneath membrane.
b. The dry paper will wick the water through the gel. The water takes the DNA with it. The DNA is
transferred from the gel to the solid support membrane.
c. This same procedure performed with protein is known as “Western blotting”
XXIX. A single strand of DNA will stick to its complement [S25]
a. Probes with reporters are sequences of DNA that will complement with the sequence of DNA you are
looking for. The reporter could be radioactive or fluorescent. This allows us to detect where the
probe is bound.
XXX. A single strand of DNA will stick to its complement [S26]
a. Wash away the excess DNA (that’s not bound) and now we can visualize only the DNA that is bound
to the probe.
XXXI. DNA can be copied by a polymerase enzyme [S27]
a. Similar to how we naturally replicate our own DNA but it’s not exactly the same.
XXXII.
DNA can be copied by a polymerase enzyme [S28]
a. There is a pool of nucleotides, a target DNA sequence of DNA, and a DNA polymerase.
XXXIII. DNA can be copied by a polymerase enzyme [S29]
a. The polymerase reads the anti-sense strand of DNA and pulls particular nucleotides from the media
to incorporate in the growing chain, building a complement sequence.
b. DNA is copied.
XXXIV. DNA can be copied by a polymerase enzyme [S30]
CLASS: 11-12
Scribe: Jordan Taylor
DATE: 9-22-2010
Proof: Lee Redditt
PROFESSOR: Pittler
LECTURE TITLE
Page 4 of 4
a. We can label particular nucleotides and recognize where they were incorporated into the strand.
b. We can also supply the media with dideoxy-nucleotides, which terminate the strand upon
incorporation. (these ntd’s lack a 3’ OH that’s required for chain elongation)
c. This is the basis for DNA sequencing
d. I’m Ron Burgundy?
XXXV.
DNA can be copied by a polymerase enzyme [S31]
a. every time a “T” is needed a dideoxy-nt will occasionally be added and we note an array of different
sized strands of DNA, all ending in dideoxy-“T”
XXXVI.
DNA can be copied by a polymerase enzyme [S32, S33]
a. This is a result of DNA sequencing. The bands represent frequency. Read the dnt’s straight across
to determine the sequence of that DNA chain.
XXXVII. DNA can be copied by a polymerase enzyme [S34]
a. Nobel Prize by Kary Mullis, (fun fact: credits this discovery to LSD, and believes AIDS is a conspiracy)
b. PCR = Polymerase Chain Reaction
c. We can amplify DNA into a large (therefore analyzable) quantity starting from a small quantity
d. We can actually start with one molecule and amplify up to 10^7 copies
e. We don’t have to isolate a particular region of the genome first, we can target that sequence in PCR
and only amplify that sequence
XXXVIII. DNA can be copied by a polymerase enzyme [S35]
a. The first step is denaturation = 1 minute, strands separate
b. Add primers (forward and reverse, usually 20-25 bp), one primer will bind to the sense strand, one will
bind to the anti-sense strand. Use primers specific to your DNA region of interest.
c. Annealing, primers will hybridize with DNA strands
d. Elongation – DNA polymerase pulls nucleotides and adds them to the primer strand based on the
parent strand sequence
XXXIX.
DNA can be copied by a polymerase enzyme [S36-S38]
a. The first amplification is linear, but any amplification after that is exponential
b. Theoretically, it should progress exponentially at 2^n, but this isn’t quite achieved in practice
c. The body performs closer to the theoretical ideal than the test tube reaction
XL. DNA can be copied by a polymerase enzyme [S39]
a. The Taq polymerase is used because it does not denature at the high temperatures of PCR.
b. It is found in a bacteria, Thermas aquaticus, in hot springs
XLI. DNA can be copied by a polymerase enzyme [S40]
a. The DNA sample can come from many different places
b. It doesn’t take much quantity to start with
c. PCR is often used in forensics
d. The beauty of PCR is its sensitivity, the problem with PCR is also its sensitivity
e. Thus, contamination is a significant problem
XLII. DNA can be copied by a polymerase enzyme [S41]
[End 46:47 min]