X Molecular Techniques2 Dcom Exam2 Mfm

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Molecular cloning allows for the creation of two types of libraries. the 2 types of libraries are ... and ...

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genomic DNA library
cDNA library

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this DNA library represents expressed genes only:

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cDNA

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the construct the genomic library, genomic DNA is partially digested into segments. the segments must represent ... and each segment must ... another genomic fragment in the library

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the entire genome
overlap

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in constructing a cDNA library, the source is total ... from a specific tissue or developmental stage of embryogenesis

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RNA

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in creating a cDNA library, after extraction of total RNA, the next step is to ...

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Isolate the polyadenylated mRNA

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in constructing a cDNA library, after isolating the polyadenylated mRNA, you will need to use ... to convert the mRNA into ds cDNA

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reverse transcriptase

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in constructing a cDNA library, after you have you double-stranded cDNA, you will want to do what with it?

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Clone the cDNAs into vectors

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what is so special about a vector that contains a Shine Delgarno sequence?

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it is an expression vector that will express the protein being cloned

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In a cDNA library, the genes transcribed at a ... frequency will be represented more often than those transcribed at a lower frequency.

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higher

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... are short sequences of single stranded DNA that are complimentary to DNA sequences of specific alleles and can be radioactively labeled and used as probes to detect single nucleotide changes.

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Allele-specific oligonucleotides (ASO)

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Any one of a series of two or more different genes that occupy the same position (locus) on a chromosome.

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allele

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what is the advantage of Allele-specific oligonucleotides (ASO) analysis over Southern blot analysis?

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ability to detect single nucleotide changes

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RFLP stands for ...

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Restriction Fragment Length Polymorphism

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the following describes what:
Genetic variations at the site where a restriction enzyme cuts a piece of DNA. Such variations affect the size of the resulting fragments. These sequences can be used as markers on physical maps and linkage maps.

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RFLP

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A common variation in the sequence of DNA among individuals.

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polymorphism

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there are 2 types of RFLP's. briefly describe each:

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1. mutation leads to creation or deletion of restriction site

2. Alteration of genetic material leads to change in size of restriction site

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what is an indel?

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a mutation that inserts or deletes nucleotides into/from a sequence

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what is a point mutation?

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a mutation that replaces one of the nucleotides

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the following describes what:
the synthesis of new DNA strands on a single stranded template and the random incorporation of chain-terminating nucleotide analogues.

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Sanger method

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what is needed for DNA sequencing:
1.
2.
3.
4.
5.

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1. ssDNA template
2. labeled primer
3. DNA polymerase
4. the four dNTPs
5. 4 separate reaction tubes containing one of four
dideoxynucleotides (ddNTPs) - chain terminators

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in DNA sequencing, what type of gel must be used?

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denaturing polyacrylamide gel

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... exploits highly variable repeating sequences and based on the idea that an individual will have a distinct pattern of these variable repeating sequences

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DNA fingerprinting

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VNTR stands for:

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variable number of tandem repeats

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minisatellite

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repeat is 9-65 nucleotide in length

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microsatellite

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di-, tri- or tetranucleotide repeats

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what are the basic steps for DNA fingerprinting?

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total genomic DNA digested and Southern blotted. use probe against the repetitive sequence (if there is limited amount of DNA available, PCR)

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briefly describe the microarray technique:

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isolate mRNA from cell
use this mRNA as templates to generate cDNA with a "fluorescent tag"
labeled samples are then mixed and incubated with a microarray containing the immobilized genes A, B, C, and D
labeled molecules bind to the sites on the array corresponding to the genes expressed in each cell.

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why would one use the microarray technique?

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analyze expression of many genes in a single experiment

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if you test healthy tissue vs diseased tissue using microarray, what might you tell about the diseased tissue?

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which genes are turned ON/OFF in response to disease
gene expression pattern of a disease state

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microarrays can be used for comparative genomic hybridization which is used to detect ...

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genomic loss or gain
change in the number of copies of a particular gene involved in a disease

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comparative genomic hybridization can be used to monitor mutations related to cancer progression

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false

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mutation microarray analysis can look at single nucleotide ... associated with particular disease

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polymorphisms

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introduction of a new gene into an organism refers to ...

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transgenic

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the following describes what:
Genes responsible for particular traits or disease susceptibility are chosen and extracted. Next they are injected into fertilized mouse eggs. Embryos are implanted in the uterus of a surrogate mother. The selected genes will be expressed by some of the offspring.

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transgenic mice

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what is a knockout mouse?

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genetically engineered mouse that has had one or more of its genes made inoperable

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what is the purpose of a knockout mouse?

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determine possible function of normal gene in whole animal

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... is the random insertion of mutant gene in genome and is useful when mutant gene ... function of normal gene

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gene addition
overrides

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transgenic mouse-TDD expresses defective version of ... impacting both transcription and DNA repair and mimics mutation in humans that causes ...

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XPD gene (TFIIH)
trichothiodystrophy (TDD)

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transgenic mouse-TDD suffers from ...

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premature aging

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what is the correlation between transgenic mouse-TDD what happens in humans?

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accumulation of DNA damage contributes to aging process