Tools of Molecular Genetics Flashcards by Joel Glotfelty

Cut DNA molecules at specific sites

-Reckognizes short sequences of double stranded DNA up to 4-8 bps

Restriction Nucleases

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Recognize palindromic (read the same 5’ to 3’) sequences

Restriction Nucleases

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Once restriction enzymes recognize the pallindromic sequences, they cut in which two ways?

) Blunt ends

2. ) Sticky ends

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A cut at the center of the recognition sequence results in

Blunt ends

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The sticky end cuts are very useful when we are trying to

Clone DNA

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RNA gel electrophoresis is similar to DNA gel electrophoresis with what exception?

RNA forms extensive secondary structure

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The extensive secondary structure that RNA forms prevents it from migrating strictly according to its size. How do we get around this?

Add denaturants such as formamide

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What do we use to visualize the electrophoresis gels of DNA and RNA?

Ethidium bromide staining or radioisotope labeling

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To visualize the DNA bands, the gel is soaked in a dye (such as ethidium bromide) that binds to DNA and fluoresces brightly under

UV light

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An alternative method for visualizing DNA is

-The exposure of x-ray film to a radioactive sample

Autoradiography

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DNA can be labeled with radioactive isotope

-Will expose autoradiographic film

P32

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Allows us to compare and analyze DNA and RNA molecules of identical or related sequences

Hybridization

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Advantageous for detecting specific species of DNA or RNA in a mixture and estimating the quantities of each

Hybridization

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The single stranded DNA or RNA used to detect the unknown DNA is called a

Probe

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How can we denature the double stranded DNA or folded RNA so that we can hybridize with our probe?

Denature with either High temp or High pH

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Once we add the probe, we can allow the DNA to reannel by

Lowering the temp or pH

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We pick conditions to ensure that DNA or RNA anneals with our probe. How well these conditions allow our DNA or RNA to anneal together is called

Stringency of conditions

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The higher the stringency, the

Lower the probability of hybridization

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Increasing the temperature or amount of denaturing agent such as formamide raises the

-Lowers probability of hybridization

Stringency

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If we want only a very specific fragment to anneal with a probe, we can adjust conditions accordingly. For example, we could

Raise temp or concentration of formamide

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If we want to anneal heteroduplexes with mismatches, we can use

Lower temp or lower conc. of formaldehyde

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Another way to manipulate stringency is through the length of the

Probe

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Form stable heteroduplexes with target sequences that are similar but not identical to the probe

Longer nucleic acid sequences (more than 100 nucleotides long)

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Allows cross-species analyses and identification of distantly related members of a gene family

Reduced stringency

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Less tolerant of sequence mismatches than long probes

Short probes

Makes it so that it is possible to select for perfectly matched duplexes only

Short probes (High stringency)

Long probes can form stable hybrids even in the presence of

Mismatches

Allow you to distinguish between allelic sequences that differ by just a single nucleotide

Short probes (High stringency)

An inherited difference in the pattern of restriction enzyme digetion

Restriction Fragment Length Polymorphism (RFLP)

In order to detect RFLP we need to use

Restriction fragments

The target molecule of interest in southern blotting is

DNA

Used to detect specific DNA fragments

Southern Blotting

Useful in investigating the number of copies of a gene or whether there are large deletions, insertions, or rearrangments -Can also be used to detect a point mutation

Southern Blotting

Describe a southern Blot

1. ) Unlabeled DNA cut w/ restriction enzyme 2. ) DNA fragments separated by agarose gel electrophoresis 3. ) Fragments blotted onto nitrocellulose paper 4. ) Labeled DNA probe hybridized to separated DNA 5. ) Sheet is washed so that only hybridized DNA fragments remain 6. ) Labeled hybridized fragments visualized by autordiography

An adaptation of southern blotting to detect specific sequences in RNA

Northern Blotting

The radioactive probe for Northern Blotting is usually a

Single stranded cDNA molecule

Allow us to monitor gene expression levels

Northern Blots

More efficient way than a Northern Blot to monitor gene expression levels

DNA microarrays

Used to monitor the expression of many thousands of genes simultaneously

DNA microarrays

How do DNA microarrays work?

Reverse transcribed mRNA is turned into cDNA labeled with fluorochrome and hybridized to a microarray. The colored spots on the microarray will show which gene is expressed at a higher level

Designed to match the nucleotide sequence flanking the substitution

Allele-specific Oligonucleotides (ASOs)

Patient DNA is hybridized to a panel of mutation specific probes

Allele specific Blotting

Allele specific blotting is used to detect things like

Sickle cell mutations and as a strategy for screening for thalassemias

Simple and rapid technique to amplify the sequence of a target DNA up to 10^9 fold in just a few hours

Polymerase Chain Reaction (PCR)

A valuable tool in medicine because it allows for the ampliphication of even trace amounts of DNA

PCR

PCR is a repetition of many cycles consisting of three steps. What are the three steps?

Step 1.) Heat to separate DNA strands Step 2.) Cool and add primers (annealing) Step 3.) Primers are incubated w/ DNA polymerase and the four deoxynucleotides, and complementary strands are synthesized

In order to perform PCR we need to design two

Primers (one for each strand)

Direct the amplification of the desired piece of DNA

PCR Primers

PCR depends on usage of a heat stable

Polymerase (taq polymerase)

What amplification does each cycle of the PCR have?

Each cycle doubles the number of strands from the previous cycle

Uses repeat rounds of strand separation, hybridization, and synthesis to amplify DNA

PCR

Dependent on perfect base-pairing of the 3' end of nucleotide primers

Allele specific PCR

Allows us to selectively amplify a mutant allele cause by a mismatch -because the designed primer will bind to the mismatch much better than taq polymerase

Allele specific PCR

mRNA is isolated from a tissue, and then a cDNA is synthesized using a reverse transcriptase. The original RNA template is removed by RNAse H and cDNA is amplified. PCR then occurs

Reverse Transcriptase PCR (RT-PCR)

Removes the RNA tamplate in RT-PCR

RNAse H

In this technique, a target DNA strand is replicated in vitro using a primer, DNA polymerase, and a mixture of dNTPs for adenine, guanine, thymine, and cytosine in four different reaction tubes. Each reaction tube also contains a small amount of one of the four radiolabeled nucleotide analogs

DNA Sanger sequencing

How does Sanger sequencing work?

Randomly, the radioactive nucleotide analogs will be inserted into the newly synthesized strand and synthesis will stop. This allows us to see which nucleotide occupies which position in the target DNA

The nucleotide analogues lack the

3' OH needed for synthesis to continue

When the Sanger sequencing reaction is complete, each tube contains a mixture of radioactively labeled

DNA fragments of different length

The bottom of the gel for Sanger sequencing represents the

5' End of the newly synthesized DNA

The top of the gel for Sanger sequencing represents the

3' end of the newly synthesized strand

The strand synthesized by Sanger sequencing will be complementary to our

Target strand

A single reaction tube will contain the target DNA, a primer, all four dNTPs and all four analogues

Automated DNA sequencing

In automated DNA sequencing, the PCR reaction will generate the DNA fragments that are separated by capillary electrophoresis. The fluoresence emission of each peak is monitored by a laser detector and recorded on a chromatogram where the 5' end of the sequence is on the

Left

Recombinant DNA can be copied inside of

Bacterial Cells

Some bacteria can efficiently take up foreign DNA from their surroundings, a phenomenon called

Transformation

Plasmids that are taken up by the host bacteria are maintained as a piece of DNA independent of the bacterial chromosomes. Therefore, they are useful tools because their replication is independent of the

Host Cell

Specialized plasmid vectors are used to

Clone DNA

How do we clone DNA using plasmids?

Cut open circular plasmid and insert the DNA fragment to be cloned. When the resulting recombinant DNA replicates, our fragment will be cloned

Cleaves the circular double-stranded plasmid (vector) DNA for insertion of the DNA fragment we want to clone

Restriction Nuclease

The DNA fragment we want to clone is covalently linked to the vector by

DNA ligase

The DNA fragment we wish to clone must be cut with the same

Restriction enzyme

The recombinant plasmid is made inside of a test tube. When we introduce the recombinant plasmid DNA into a bacterial cell, what happens?

The plasmid will be replicated millions of times

We can use DNA cloning by plasmid vectors to compile a

DNA library

A collection of cloned fragments of an organism - Two types 1. ) Genomic 2. ) cDNA

DNA libraries

Libraries of human genomic DNA fragments can be constructed using

Restriction nuclease and ligase

A bacterial colony carrying a particular DNA clone can be identified by

Hybridization

When compiling a genomic library, once we have transferred our bacterial colonies to a sheet, we can lyse the bacteria and denature the DNA with

Alkali

After we have denatured the DNA, we then add radioactively labeled DNA probes specific for the plasmid of interest. Then we expose the paper to

Photographic film

Living cells containing the plasmid of interest can then be isolated from the colony disk and

Grown in large quantities

Represent the mRNA produced by a particular tissue

cDNA libraries c = complimentary

cDNA is double stranded DNA synthesized from RNA by

Reverse transcriptase and DNA polymerase

Genomic DNA clones and cDNA clones derived from the same region of DNA are

Different

Exons, introns, and non-transcribed DNA are included in the DNA clones of the

Genomic Library

The intron sequences are removed and a continuous coding region is present in each clone of the

cDNA library

Allows you to see which genes are expressed more frequently

cDNA Library

Genes will be represented equally regardless of their levels of expression in the

Genomic Library

Can be used to splice together a set of DNA fragments derived from different sources (making Chimeras)

Serial DNA cloning

Why do we need to make chimeric proteins?

Adding a fluorescent protein to a protein of interest allows us to visualize where the protein is located in the cell

In serial DNA cloning, after each DNA insertion step, the recombinant DNA is

Cloned (purifies sample)

The purified cloned DNA is then cut with a restriction nuclease, and

Another DNA fragment is added

Can be produced from a protein-coding sequence cloned into and expression vector and introduced into cells

Large amounts of protein

To use DNA cloning to make a protein, we first must make a vector plasmid containing a

Highly active promoter for the protein of interest

Make it possible to move experimentally from gene to protein and from protein to gene

Recombinant DNA techniques

If we want to make DNA from a protein, we must determine the amino acid sequence of a purified peptide fragment, then we want to

Search the DNA database for the gene sequence

Used to determine the pattern of a gene's expression

Reporter Genes

Let's say we want to find out which cell types express protein "X" but it is difficult to detect the protein "X" directly, what can we do?

Replace the coding sequence for protein "X" with a reporter gene that expresses a protein like GFP which can be easily monitored

The reporter protein will only be expressed in places where

The target protein is expressed

In order to tell which regulatory regions control expression in particular cell types, we

Turn off all but one sequence at a time and see where the protein is expressed

We can add epitopes as

Reporters

In order to create an epitope tagged protein, we fuse the gene for the epitope tag to the gene for the

Target Protein

We can use DNA cloning and recombinant DNA technologies to genetically modify animals by

Gene replacement, gene knockout, and gene addition

When the normal gene is entirely replaced by a mutant copy of the gene -provides information on the activity of the mutant gene without interference from the normal gene

Gene replacement

The normal gene can be completely inactivated by making large deletions in it. This process is called -Widely used to obtain information on the function of the normal gene

Gene knockout

A mutant gene can be added to the normal genome. This provides information when the introduced mutant overrides the function of the normal gene

Gene addition