detecting mutations- lecture 4 Flashcards
pcr primer design
-must have 2 primers, each complementary to a different dna strand
-2 primers must flank sequence of interest, but doesnt need to be precise
how does pcr work
-heat dna to separate 2 strands
-cool down and add primers to anneal to complementary sequences
-reheat and add dna polymerase to create 2 new double stranded dna molecules
—the entire cycle repeated and each time the amount of target dna doubles
what to do with the piece of amplified dna
run a gel
genotype
the set of genes possessed by an individual organism
phenotype
appearance or manifestation of a characteristic in a given organism
allele
a particular version of a gene
locus
a specified position on a chromosome, used more or less interchangeably with gene
what are restriction enzymes
restriction enzymes provide bacteria against invading viruses but cutting up the foreign dna when it enters the cell
host dna is protected from cutting by being enzymatically modified in such a way as to prevent dna cleavage
how does gel electrophoresis separate dna fragments
according to their size- larger fragments move slower through the gel
amyotrophic latera sclerosis, als
neurological decay disease
dominant allele
the outcome is the same regardless of whether the allele is homozygous or heterozygous
gain of function mutation
altered in some way the behavior of the allele
haploinsufficiency
The situation that occurs when one copy of a gene is inactivated or deleted and the remaining functional copy of the gene is not adequate to produce the needed gene product to preserve normal function
recessive allele
trait will only be expressed if an individual is homozygous for the mutant allele
ivf
introducing egg to sperm in petri dish
A term used to describe a gene when it is found in its natural, non-mutated (unchanged) form
wild type allele
how does sanger sequencing work
DNA sequencing is
the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine
homozygous
Individuals who are homozygous have 2 of the same allele. That can be either AA or aa, homozygous dominant or homozygous recessive
homozygous dominant
An individual who is homozygous dominant for a particular gene will have two dominant “A” alleles for that gene
homozygous recessive
An individual who is homozygous recessive will have the “aa” genotype, two recessive “a” alleles
heterozygous
Individuals who are heterozygous have two different alleles for a particular gene. Typically, this is represented as “Aa”
sporadic
no prior family history
familial
runs in the family
What is a chromosome?
Chromosomes are thread-like structures located inside the nucleus of animal and plant cells. Each chromosome is made of protein and a single molecule of deoxyribonucleic acid (DNA). Passed from parents to offspring, DNA contains the specific instructions that make each type of living creature unique
What do chromosomes do?
The unique structure of chromosomes keeps DNA tightly wrapped around spool-like proteins, called histones. Without such packaging, DNA molecules would be too long to fit inside cells.
For an organism to grow and function properly, cells must constantly divide to produce new cells to replace old, worn-out cells. During cell division, it is essential that DNA remains intact and evenly distributed among cells. Chromosomes are a key part of the process that ensures DNA is accurately copied and distributed in the vast majority of cell divisions.
Changes in the number or structure of chromosomes in new cells may lead to serious problems. For example, in humans, one type of leukemia and some other cancers are caused by defective chromosomes made up of joined pieces of broken chromosomes.
It is also crucial that reproductive cells, such as eggs and sperm, contain the right number of chromosomes and that those chromosomes have the correct structure. If not, the resulting offspring may fail to develop properly. For example, people with Down syndrome have three copies of chromosome 21, instead of the two copies found in other people
The only human cells that do not contain pairs of chromosomes
reproductive cells, or gametes, which carry just one copy of each chromosome. When two reproductive cells unite, they become a single cell (zygote) that contains two copies of each chromosome. This cell then divides and its successors divide numerous times, eventually producing a mature individual with a full set of paired chromosomes in virtually all of its cells
What are centromeres?
The constricted region of linear chromosomes is known as the centromere. Although this constriction is called the centromere, it usually is not located exactly in the center of the chromosome and, in some cases, is located almost at the chromosome’s end. The regions on either side of the centromere are referred to as the chromosome’s arms.
Centromeres help to keep chromosomes properly aligned during the complex process of cell division. As chromosomes are copied in preparation for production of a new cell, the centromere serves as an attachment site for the two halves of each replicated chromosome, known as sister chromatids
What are telomeres?
Telomeres are repetitive stretches of DNA located at the ends of linear chromosomes. They protect the ends of chromosomes in a manner similar to the way the tips of shoelaces keep them from unraveling.
In many types of cells, telomeres lose a bit of their DNA every time a cell divides. Eventually, when all of the telomere DNA is gone, the cell cannot replicate and dies.
White blood cells and other cell types with the capacity to divide very frequently have a special enzyme that prevents their chromosomes from losing their telomeres. Because they retain their telomeres, such cells generally live longer than other cells.
Telomeres also play a role in cancer. The chromosomes of malignant cells usually do not lose their telomeres, helping to fuel the uncontrolled growth that makes cancer so devastating.
what can repeats of dna sequence cause
huntington’s disease
Polymerase Chain Reaction (PCR)
PCR can be used to isolate and amplify a specific DNA sequence from a sample. For example, it can be used to selectively amplify the sequence of a particular gene from an individual’s genome
The main components of a pcr reaction are
Template DNA, Primers, Taq DNA polymerase, dNTPs
Template DNA
genomic DNA from the individual to be tested
Primers
short fragments of DNA that determine the “boundaries” of the sequence to be copied; each primer serves as the starting point for synthesis of a new DNA strand
Taq DNA polymerase
a DNA polymerase that can withstand high
temperatures without becoming denatured. It synthesizes the new DNA strands
dNTPs
deoxynucleotides, the building blocks of DNA, which will be used to synthesize the new strands
In performing PCR, researchers combine these components in a small tube, then subject the mixture to repeated cycles of heating and cooling to allow exponential amplification of the DNA. A typical PCR
cycle consists of
- 30 seconds at 94°C. DNA is denatured into single strands.
- 30 seconds at the annealing temperature for the particular primer pair used in the reaction (often 50-65°C). At the annealing temperature, strands of DNA anneal (hybridize). Because the primers are present at much higher concentrations than the template DNA,
the template DNA will anneal to the primers instead of reforming its original double helix. - 30 seconds at 72°C. Taq polymerase extends the primers, resulting in a new strand of DNA
The results of a PCR (the PCR products) are typically analyzed using
gel electrophoresis
The PCR products are loaded into a well at the end
of an
agarose gel in a buffered salt solution
how does Gel Electrophoresis work
The PCR products are loaded into a well at the end
of an agarose gel in a buffered salt solution. An electric current is then applied across the gel. DNA is negatively charged due to its phosphate backbone, so it is pulled through the gel towards the positive electrode. As it travels, DNA fragments of different sizes are separated, because they move through the gel at different rates. Smaller fragments move more quickly through the gel, and larger fragments more slowly. As a consequence, when you stop the current, larger fragments will be closer to the wells, and smaller fragments
closer to the other end. The fragments can be visualized using a DNAbinding dye. Fragments of a particular length will appear as a discrete
band in the gel
PCR is a very powerful molecular biology technique, but it gives you a limited amount of information. What can you determine?
if an individual’s genome contains a given sequence. If the primers bind and produce a PCR product, the sequence is present; if they are unable to bind, no
product is produced, and the sequence is likely absent. You can also determine the length of the sequence between the primers. However, you cannot determine the specific sequence of bases in the PCR product. Thus, gel electrophoresis can be used to detect only
those mutations that result in a detectable change in the length of a gene or sequence, generally between tens and a few thousand bases. Mutations that can therefore be detected by PCR include trinucleotide
repeat expansions, transposon insertions, and other insertions and deletions of 10’s-1000’s of bases
Restriction Fragment Length Polymorphism (RFLP)
a molecular biology technique that you can use to detect a subset of changes to DNA sequences that affect one or a few bases, such as a single base substitution
In order for this type of analysis (rflp) to be applicable
for the detection of a particular sequence variant
it needs to either create or interrupt a restriction site in the DNA sequence
Restriction sites are specific short DNA sequences that are recognized and cut by
restriction enzymes/restriction endonucleases
restriction enzymes/restriction endonucleases
found in bacteria, where they serve as a defense mechanism against viruses. The bacteria use restriction enzymes to cut up viral DNA, preventing an infection from progressing and the virus from replicating.
Today, biotechnology companies mass produce restriction enzymes for use in molecular biology labs, where they are commonly used to manipulate DNA.
Each restriction enzyme recognizes a specific DNA sequence, and cuts that sequence between specific bases. These restriction sites are generally between four and eight bases long, and many are palindromic
– the sequence on one strand, as read in the 5’ to 3’ direction, is the same as the sequence on the other strand in the 5’ to 3’ direction.
Note: a single base change is sufficient to stop EcoRI
from recognizing the site
The general procedure for RFLP analysis involves three different molecular biology techniques
- PCR to isolate and amplify the region of interest
- Restriction digestion with an enzyme that would cut one version of the gene, but not the other
- Gel electrophoresis to analyze the results
Sanger Sequencing
The Sanger method uses the same enzyme that copies DNA naturally in cells, DNA polymerase. The trick involves making the copy out of base pairs that have been slightly altered. Instead of using only the
normal “deoxy” bases (As, Ts, Gs, and Cs) found naturally in DNA, Sanger also added some so-called “dideoxy bases.” Dideoxy bases have a
peculiar property: DNA polymerase will happily incorporate them into the growing DNA chain (i.e., the copy being assembled as the complement
of the template strand), but it cannot then add any further bases to the chain. In other words, the duplicate chain cannot be extended beyond a dideoxy base
Next-generation sequencing
is an umbrella term used to describe a
number of different modern sequencing technologies. PCR-based sequencing has been in use for almost 40 years. However, this older method of sequencing was limited in the amount of data it could generate. Next-generation methods have many advantages: they have
made sequencing hundreds of times faster, far less expensive, and are able to generate unprecedented amounts of data. These advances have revolutionized the field of genetics and genomics and are leading to
major breakthroughs in the understanding and treatment of disease. The end result of a typical next gen sequencing run is many copies of small overlapping fragments of DNA. These copies are referred to as ‘reads’. For example, a single sequencing reaction using the industry standard Illumina technology, can produce between 50 million and 1
billion reads, depending on which machine performs the sequencing. In order to make sense of such massive amounts of data, computational
tools have been developed to process all this information.
phenotype
is their appearance or the way in which they
manifest a particular characteristic
genotype
An individual’s phenotype is often
determined by their genetic makeup
alleles
2 versions of a gene
Individuals that have two different alleles of a gene are said to be
heterozygous for that gene
those that have two copies of the same
allele are
homozygous for that gene
If an individual is heterozygous for a particular gene, they will express
the phenotype associated with one of their two alleles. The phenotype that they express is said to be dominant. Individuals who are
homozygous for the dominant allele or heterozygous will, for simple Mendelian traits, display the same phenotype.
individuals must be homozygous in order to display the phenotype for a
recessive trait
loss-of-function (also
known as null) mutations
Many recessive traits are due to what we call loss-of-function (also known as null) mutations. These types of mutations cause a gene to lose its ability to perform its normal function. These mutations are often
recessive because having a single copy of the normal allele is sufficient for function. However, some loss-of-function mutations cause even heterozygous individuals to exhibit the mutant phenotype. Genes for
which a loss-of-function mutation produces a phenotype are said to display haploinsufficiency: losing the function of one of two copies causes
a phenotype. Mutations in these genes will show a dominant inheritance pattern
Many dominant traits are due to gain-of-function (also known as neomorphic) mutations. These mutations cause genes to…
do or create something new that it normally would not- the gene has a new function.
Regulatory mutation
mutation is in a DNA sequence that is involved in
regulating gene expression, such as an enhancer, promoter, splice site, or terminator, etc
Coding mutation
mutation is in the body of the gene that codes for the
protein
Base-pair substitution
one base pair in a DNA duplex is replaced with
another
Insertion or deletion
one or more extra or missing nucleotides
Frameshift
insertion or deletion that involves a number of bases that is not divisible by three; shifts the triplet code for the gene “out of phase,” leading to a completely different amino acid sequence after
the mutation
Simple Sequence Repeats
Simple sequence repeats (SSRs), sometimes described as genetic ‘stutters,’ are DNA tracts in which a short base-pair motif is repeated several to many times in tandem (e.g. CAGCAGCAG). These sequences experience frequent mutations that alter the number of repeats.
Preimplantation Genetic Diagnosis (PGD)
PGD involves testing an embryo before it implants for a specific, known genetic disorder. PGD is used so that embryos unaffected by the disorder can be returned to the uterus.
coding vs noncoding rna
Coding RNAs generally refers to mRNA that encodes protein to act as various components including enzymes, cell structures, and signal transductors. Noncoding RNAs act as cellular regulators without encoding proteins
These novel appearances of a disease are termed
sporadic
