Biotechnology Flashcards
(95 cards)
1
Q
What are the 4 achievements leading to modern molecular biology?
A
- Restriction Endonucleases (enzymes)
- Cloning DNA
- Creation of Synthetic Probes
- PCR
2
Q
What are restriction endonucleases?
A
Restriction endonucleases are enzymes that split very specific DNA sequences.
3
Q
Restriction endonuclease sequences are usually very ____ and are generally ______ which are read 5’ to 3’.
A
short
palindromes
4
Q
Restriction enzymes cleave to leave either ____ or ends or ____ ends.
A
sticky or blunt ends
5
Q
The ends are jagged for ___ ends.
A
sticky ends
6
Q
The ends have no base pair overlap for ___ ends.
A
blunt ends
7
Q
The 3’ OH group and the 5’ phosphate are attached after cleavage which is important for _____.
A
ligation.
8
Q
A DNA sequence that can be cleaved by a restriction enzyme is called _______.
A
restriction site
9
Q
Restriction enzymes that recognize larger sequences cut ____ frequently.
A
less
10
Q
Restriction enzymes are tools to ____, _____, and _____ DNA.
A
cut, paste, and analyze DNA
11
Q
Depending on how many time a base recognizes a sequence determines how it ____ it.
A
cuts
12
Q
DNA Cloning:
What is recombinant DNA?
A
Recombinant DNA is when fragments of DNA can be “pasted” together to make hybrid molecules.
13
Q
DNA cloning is very easy with ____ ends.
A
sticky ends
14
Q
DNA Cloning
DNA _____ is the enzyme which creates the phosphodiester bonds.
A
ligase
15
Q
DNA Cloning
DNA cloning involves inserting a ____ into a cloning vector.
A
restriction fragment
16
Q
DNA Cloning
A vector can then be replicated in host cells (usually bacteria, sometimes yeast or other cells). DNA is now amplified. This is called ________.
A
Recombinant DNA Amplification
17
Q
DNA Cloning
You can recognize where DNA fragments are joined together based on the ___________.
A
palindrome sequence
18
Q
DNA Cloning
Molecules of DNA that can accept fragments of foreign DNA are called _________.
A
vectors
19
Q
DNA Cloning
Vectors must be capable of _____of the cell. It must have at least one restriction site for foreign DNA insertion. Vectors must carry at least one gene for selection (usually antibiotic resistance).
A
autonomous replication
20
Q
DNA Cloning
What are the most common vectors?
A
Prokaryotic plasmids
21
Q
What are other vectors besides plasmids?
A
- Phages
- Yeast plasmids
- Yeast Artificial Chromosomes (YACs)
- Mammalian virueses (i.e. the retrovirus)
22
Q
What are the two types of DNA libraries?
A
Genomic DNA Libraries
cDNA Libraries
23
Q
How are genomic DNA libraries made?
A
The entire genome is chopped up with restriction enzymes, cloned directly to vectors, and used to transform bacteria.
Each transformed bacteria containing a plasmid may contain a different segment of the genome (thousands are collected to assure the whole genome is represented).
24
Q
What do genomic DNA libraries represent?
A
The collection contains ALL sequences of the genome, including coding regions, introns, promoters, and intervening sequences.
25
How can genomic DNA libraries be used?
If you have a good enough section of bacteria cells that amplifies the DNA and you collect the DNA from them (the entire library), each one of the samples that you saved should represent the entire genome of the original host.
26
What do cDNA libraries represent?
cDNA is not representative of the entire genome. cDNA represents the expression pattern at the time the sample was collected.
**Ideally, a cDNA library will contain sequences representing all mRNAs present in the cell or tissue type at the time the mRNA was collected.
27
How can cDNA libraries be used?
cDNA allows one to see what genes were being expressed in a particular cell or tissue type.
***** cDNA only contains mRNA sequences!!!!! There are NO introns, promoters, etc.
28
How are cDNA libraries made?
cDNA is generated using isolated mRNA from a particular cell or tissue type.
The mRNA is reverse transcribed (by reverse transcriptase) and the second strand is synthesized using a DNA polymerase.
cDNA is ligated into a vector, used to transform bacteria and 1000s of clones are collected.
29
DNA from a cDNA library can be cloned into an expression vector for _______ production.
protein production
30
What are used to transform bacterial strain expression?
1. Promoter
2. Shine-Dalgarno sequences (used to know where to start translation)
3. cDNA
31
What is DNA sequencing used to determine?
It is a technique used to determine the exact sequence of:
1. Cloned fragment
2. PCR amplified stretch of DNA
32
DNA Sequencing
DNA is melted to generate a:
single-stranded template
33
DNA Sequencing
What are the major components of the reaction used to conduct DNA sequencing?
DNA + dNTPs + DNA primer + polymerase
34
DNA Sequencing
In DNA sequencing, the sample is split into 4 tubes. Each tube contains a small amount of a specific dideoxyribonucleotide and they are:
ddCTP
ddATP
ddGTP
ddTTP
35
DNA Sequencing
Important: What stops elongation?
ddNTPs
36
What is a probe?
A probe is a single stranded DNA (ssDNA) that is complimentary.
37
What is hybridization?
A target DNA is made single-stranded (heat, chemicals)
The target is immobilized on a solid support (like nitrocellulose membrane) so it cannot re-anneal with its original complementary strand.
The ssDNA-coated membrane is exposed to a probe.
If a complementary sequence is present, a probe will bind the immobilized ssDNA and can be identified via audioradiography.
38
What happens first in hybridization?
A target DNA is made single-stranded (heat, chemicals)
39
Step 2 in hybridization:
The target is immobilized on a solid support (like _________) so it cannot re-anneal with its original complementary strand.
nitrocellulose membrane
40
Step 3 in hybridization: The ssDNA-coated membrane is exposed to a _______.
probe
41
Step 4 in hybridization: If a complementary sequence is present, a probe will bind the _____ ssDNA and can be identified via ________.
immobilized
audioradiography
42
What are the two types of probes?
Smaller and Larger
43
What is the importance of ddNTP?
At the 2’ carbon that is where the deoxy forms and at the 3’ that is where the –OH is; if you take that and make it deoxy, you have a ddNTP. ddNTP does not have OH; chain elongation stops once that is inserted; this generates the nucleotide sequence à NTPs.
44
Smaller probes- chemically synthesized ______ (2-30 bases); this is the same way synthetic primers are made; smaller probes can be VERY _____, i.e. identify a single base mutation in a sequence; just 1 nucleotide difference will inhibit the ability of the probe to bind to the DNA strand thought to be the region of interest. Small probes are used for _______reasons.
oligonucleotides
specific
diagnostic
45
Larger probes- made via one of several molecular biology techniques (i.e., _______, _______, and several others); much less _____- can be used to identify similar genes in different organisms or the same gene in different individuals that may not be exactly the same in sequence; used less for diagnostic reasons and more for ________ reasons, as well as genomic fingerprints; it is more comparative.
reverse transcription, PCR,
specific
hereditary, ancestry
46
Southern blotting: analysis of _____.
DNA
47
Southern blotting: analysis of DNA
o DNA is isolated and subject to _______
o Digested DNA is then subjected to __________
o DNA is denatured, blotted (______onto a membrane)
o Blot (membrane) is ______.
restriction digestion
gel electrophoresis
immobilized
probed
48
Northern blotting: analysis of RNA (mRNA)
o Do not need to make single stranded because RNA is already single stranded; this is not used by choice.
o Probe must be complementary to _____
o Only detects ____ sequences.
o Can be used for tissue or cell specific studies.
o Is quantitative (to measure gene expression).
o It is important to realize that the probe recognizes the presence of something being expressed, as well as how much is being expressed.
mRNA
expressed
49
Western blotting: analysis of Protein
o Probe is an _______ specific to the protein of interest, usually attached to an _____ to identify positive reaction on a blot.
o How much protein is present determines how much of the antibody is bound to the _____ (protein of interest).
o Also, is quantitative and quantitative.
antibody
enzyme
antigen
50
Restriction Fragment Length Polymorphisms (RFLPs)
o Humans (not related) – 1 in 1,500 nucleotides is different (0.1%)
o Genetic differences due to ____ or _____.
o Only a few percent of the genome (exons) actually codes for proteins.
o Most of the genome is various types of intervening sequences.
mutations or polymorphisms
51
Restriction Fragment Length Polymorphisms (RFLPs)
o Genetic variations in intervening sequences are generally not harmful, since they have no consequence on the expression of genes (proteins).
o No _______ for genetic changes in intervening sequences, therefore these regions tend to be ______.
o Genetic variations in non-coding regions with no disease are called _____. Simply a change; not harmful.
o The term “______” is usually reserved for genetic changes that cause disease.
selection pressure
hypervariable
polymorphisms
mutation
52
Restriction Fragment Length Polymorphisms (RFLPs)
Realize that the ____ strands of DNA migrate through the electrophoresis gel faster, while _____ strands migrate slower. Also, realize that the human genome varies among humans most of the differences occur in regions that are not stressed to be expressed.
shorter
longer
53
IMPORTANT:
Restriction Fragment Length Polymorphisms (RFLPs)
IF a genetic change in a polymorphic region:
o 1) creates or deletes a _______
o 2) has more or less of a _____ sequence.
o Then, a RFLP is present.
restriction site
repeated
54
Restriction Fragment Length Polymorphisms (RFLPs)
| DNA variations resulting in RFLPs are
SNPs and VNTRs
55
Restriction Fragment Length Polymorphisms (RFLPs)
o 1. Single nucleotides polymorphisms (SNPs):
* single nucleotide changes
* account for 90% of genetic variation
* May ____ or ____ a restriction site
* Occasionally an RFLP is created by a disease causing mutation
* More often it is a harmless change that results in a different restriction pattern
create or abolish
56
An SNP is just a change in the sequence -one nucleotide change. But an SNP that creates RFLP affects __________.
restriction sites
57
Variable Number of Tandem Repeats (VNTRs):
* Human genome contains many regions where a sequence (2-~50 bp) is repeated in _____ many times.
* Varies greatly from person to person (not-related) and is unique for an individual.
* If DNA is cleaved on either side of a VNTR, an ____ is produced
tandem
RFLP
58
* RFLPS:
o Based on SNPs -> creation or abolishment of a _______;
Different size RFLPs based on different _____
Used to mark _________, disease markers
restriction site
restriction cutting
genes (alleles),
59
o Based on VNTRs -> more or less of a tandem repeat;
VNTRs have an expansion or non-expansion between 2 restriction sites
Different size RFLPs based on the different number of ______;
Used as molecular _____ (identifying individuals)
repeats
fingerprints
60
Compare & Contrast: DNA Cloning with PCR:
o DNA Cloning- amplify fragments by inserting them into a ____ and the host replicating the ______
o PCR- amplification done completely in a _____
* PCR revolutionized molecular biology! à Nobel prize for Kary Mullis
vector
vector
tube
61
* Primers for PCR: (Note: Primers are very similar to probes.)
o Primer design and construction – we need to know the sequence of two small flanking regions of the sequence we wish to amplify
o Primers are synthesized (~20 nucleotides) that are complementary to the ______
o Primers act as a “primer” for ______. Know that the primer itself is made up of DNA.
flanking regions
DNA polymerase
62
* PCR: (Note: PCR is a temperature-______process.)
Step 1.
o Denature the target DNA – produce ______ DNA (will allow the primers to bind when cooled)
o Heat is used to denature the DNA.
This step makes it so the primers can find a site to bind.
dependent
single stranded
63
PCR:
Step 2.
o _____the primers- DNA sample is cooled and the primers bind to the complementary sequence in the target DNA
Anneal
So, when you drop the temperature because the primers are shorter than the complementary strand of the DNA of interest, basically the primers are going to lay down first and base pair with the DNA of interest in the flanking region.
64
PCR:
Step 3
o Chain extension- a DNA polymerase uses ______ to extend the primer and build a complementary copy of the DNA strand
dNTPs
65
o Steps 1-3 repeated 20-30 times;
(Denature, Anneal, Extend) x 30
o Denaturation: ~95 degrees C
o Annealing: ~55 degrees C (When you drop the temperature, the primers will base pair with DNA of interest in the correct region.)
o Extension: ~72 degrees C
o We use heat stable __________ for PCR
(Note: “We will not be asked to remember the temperatures, but realize that they are high temperatures being used.)
Taq DNA polymerases
66
Name Advantages of PCR:
o Sensitivity: only need trace amounts of DNA (single cell)
o Speed: only takes a few hours (cloning takes a few weeks)
o DNA can be used for gel electrophoresis, Southern blotting, sequencing, etc…
o Some uses- mutation detection, detection of latent viruses (HIV), forensics, prenatal genetic diagnosis
* Note: A limitation for Northern Blot is that we are only looking at one gene at a time.
67
```
Analysis of Gene Expression:
o mRNA levels- Northern blot
* Quantitative as well as qualitative
* Look at expression of a specific gene (depending on the probe)
* Only one ______ at a time
```
gene
68
Analysis of Gene Expression:
o mRNA levels- Microarrays
* microarrays- contain _____ of immobilized sequences (probes) on glass slides
* used to compare “_____” gene expression changes in different cell types
* synthesize and label _____ from two different cell types with different _____ probes
* analyzed by a computer
thousands
global
cDNA
fluorescent
69
Analysis of Gene Expression:
o Proteins- ________- similar to genomics but looks at all the different proteins produced in particular cell or tissue types (including post-translational modifications, enzyme modulation by phosphorylation, etc…)
proteomics
70
```
Analysis of Gene Expression:
o Proteins- Enzyme-linked immunosorbent assays (ELISAs) (Hinted in class to know about ELISA! It is very important for determining something very quickly.)
```
* ______is bound to the well of a microtiter plate.
* “Probed” with an antibody linked to an _____.
* Detect by adding substrate for enzyme to form a ______.
Antigen
enzyme
colored reaction
71
Analysis of Gene Expression:
o Proteins- Enzyme-linked immunosorbent assays (ELISAs)
* Sometimes, there is a false _______.
* If it is negative, however, you can say with confidence that it is not present.
* With a false positive, follow through with the _____ Blot (specific but less sensitive.)
positive
Western
72
Remember that with protein analysis, you can use ____, _____, or ________.
proteomics, ELISA, or Western Blot.
73
Proteins- Western blot
* Protein samples are separated by gel electrophoresis based on ____.
* Proteins are blotted onto membrane.
* Membrane is probed with an enzyme-linked ____ to identify bands.
* More specific than ELISA, but more time and labor sensitive, less sensitive.
size
antibody
74
Genetic Testing- Sickle Cell Anemia:
o Sickle cell mutation in the Beta-globin gene eliminates a restriction site for ______
o Creates an RFLP: in this case, the disease-causing mutation creates an RFLP that can be used for diagnosis.
o In most diseases, an RFLP used for diagnosis is linked to the disease, but not the actual disease causing mutation. RFLP is associated with the disease because typically you have enough DNA being carried around you have a passenger _____being handed down.
o _____presence is causing sickle cell event to occur.
Restriction Enzyme MstII
polymorphism
Valine’s
75
Details of a portion of the Beta-Globin Gene:
o Southern blot with probe to beta-globin gene.
o Normal = smaller band (____ bp) ; Sickle = larger band (____ bp)
1150
1350
76
* Genetic Testing: Could do a similar procedure using PCR – amplify the region containing the potential mutation, digest PCR products with ____. This was the original work describing PCR.
MstII
77
Details of a portion of the Beta-Globin Gene:
* PCR = use primers flanking beta globin gene, or region where mutation may be
* Digest PCR product with ____, run on gel .
o Normal = one smaller band
o Sickle = one larger band
MSTII
78
IMPORTANT* Genetic Testing
Diagnosis of Sickle Cell Anemia using ASO Probes
What does ASO stand for?
allele specific oligonucleotide
79
Why is ASO is generated?
ASO is generated for recognizing allele specific differences; very specific.)
80
Genetic Testing- this technique is extremely ___ and reliable, very fast, minimal complications with the technique, non-invasive, inexpensive.
specific
81
Cystic Fibrosis:
o Caused by a genetic mutation
o Results in defective _____
o Due to improper folding and assembly of _______ chloride transport protein is prematurely degraded
o Most common lethal genetic mutation in Caucasians
o Incidence 1 in 2500
o Carrier rate 1 in 25
Cl ion channeling
CFTR- cystic fibrosis transmembrane conductance regulator-
82
Cystic Fibrosis:
o Clinical diagnostic hallmark- elevated chloride levels in sweat – specific to CF (“won’t be asked this on the block exam”)
o In the lungs, lack of _____ secretion leads to dehydration of the mucus. (Realize that there are lung issues and there is a lack of chloride secretion.)
o Mucus accumulates in the lungs, obstructs airway passages.
o Predisposed to recurrent lung infections.
o Historically, death often occurred in childhood, patients rarely survive beyond the age of 30.
o Also, causes pancreatic enzymes insufficiency and poor nutrition among other problems.
chloride
83
o Genetic testing ~70% of cases of cystic fibrosis are due to a ______ in the CFTR gene that leads to a missing ______ residue (CFTR delta F508) (“Will not be held accountable for the % of cases.”)
3-nucleotide deletion
phenylalanine
84
* PCR using primers specific to the region of the deletion-______ is only a portion of the gene.
* Other known mutations causing CF would require different analysis.
* For example: other ASO probes, other primers with PCR followed by DNA sequencing.
PCR product
85
* Genetic Diagnosis- Sickle Cell Anemia and Cystic Fibrosis
o Very specific mutations- ____ nucleotide change in sickle cell anemia and ____ nucleotide deletion in CF – ASO probes and PCR
o Some diseases have many types of mutations associated with them
single for sickle
3 for CF
86
* PKU- In USA, all newborns are screened for PKU- blood test for elevated _____ levels and low levels of ____; PKU has 400 different known mutations. (PKU converts phenylalanine à tyrosine.)
phenylalanine (high)
tyrosine (low)
87
PKU:
o Mutations can occur in any of the ____.
o Majority are ____, some splice, nonsense, insertion, and deletion mutations.
o Also notice the huge introns!
o Can we use ASO probes or PCR (directly)?
13 exons
missense
88
* PKU- To do a genetic screening for PKU, we must have:
o 1. The DNA from several family members including an affected individual
o 2. An identifiable marker associated (linked) with the particular mutation in this family (an ____)
RFLP
89
PKU
o Marker would normally be a _______
(nucleotide change that results in an added or lost restriction site)
o Generally, the marker is NOT actually the disease-causing mutation.
o Markers are at least 98% accurate in predicting the presence of a mutation.
polymorphism
90
Myotonic Dystrophy
- ________expansion disease in the 3’ non-coding region of a protein kinase gene. (Realize that the expansion is OUTSIDE of the coding region.)
o Most common form of adult muscular dystrophy.
o Because the disease is associated with repeat expansion, it will produced RFLPs when digested with a restriction enzyme on both sides of the expansion.
Trinucleotide repeat
A person effected does not have a phosphatase. Muscle can't relax. Always in a state of contraction. Muscles begin to degrade and waste.
91
PCR can be used to diagnose _____ as well- in the case of myotonic dystrophy the repeat can be so huge that PCR is difficult.
repeat expansions
92
HIV- currently, over 1 million people in the USA infected with HIV, ~25% are unaware
o Current methodology uses immunoassays for diagnosis of HIV exposure – after exposure to HIV the body produced antibodies to HIV, immunoassays uses _____ that recognize HIV antibodies
enzyme linked antibody
93
HIV
o Initial screen is using ____; a test so sensitive that false positives occur
o Second test- with Western blot
o Why not just test every sample with a Western blot?
o Blood is a huge reservoir for HIV antibodies that are present.
ELISA
94
* ______ using antibodies to HIV-antibody require that the body has mounted an immune response to a detectable level – requires a certain incubation time (~6 months)
o Can use PCR for HIV provirus (or RT-PCR/reverse transcription then PCR for HIV to test immediately)
o These tests are still being “perfected”
o Quantitative PCR is used to monitor viral load in HIV positive patients
Immunoassays
95
* Paternity Testing:
o Use _____ as molecular fingerprints
o Usually done using PCR (as opposed to traditional southern blotting)
o Legal “accuracy” differs in different states (paternity index is a measure of accuracy)
o Different _____ have a different paternity index, paternity cases always require several different _____ be used.
o PCR with primers flanking specific _____
VNTRs for all spaces
