8.9.16 Lecture

Question 1

What are restriction enzymes?

Answer 1

Restriction enzymes cleave double stranded DNA at specific nucleotide sequences.

Question 2

The nucleotide sequences at which restriction enzymes cleave are often ___.

Answer 2

Palindromic

Question 3

Restriction enzymes can cut in two ways - describe these.

Answer 3

Symmetrically (creates blunt ends) and asymmetrically (creates sticky ends, which can base pair)

Question 4

DNA fragments can be ___ to produce new combinations.

Answer 4

Ligated

Question 5

___ can join DNA fragments cut with the same restriction enzymes.

Answer 5

Base pairing

Question 6

Describe the process of creating recombinant DNA.

Answer 6

1. Restriction enzyme cuts DNA at the recognition sequence, creating 2 sticky ends.
2. DNA fragment from another source is added and fragments stick together by base pairing (annealing).
3. DNA ligase covalently seals the cut sites, creating recombinant DNA.

Question 7

A DNA fragment can be inserted into a bacterial ___ with DNA ligase.

Answer 7

Plasmid

Question 8

What is a plasmid?

Answer 8

Double-stranded DNA molecule that is separate from and can be replicated independently of chromosomal DNA.

Question 9

Describe the process of incorporating DNA into a plasmid.

Answer 9

1. Cut open the plasmid with a restriction nuclease
2. Mix the cut plasmid with the DNA fragment to be cloned. Note that this fragment is prepared with the same restriction nuclease.
3. DNA ligase and ATP are added.
4. The ends base pair and DNA ligase seals the nicks in the backbone with covalent bonds.

Question 10

Describe the process of amplification with plasmids and bacteria.

Answer 10

1. Insert the fragment of DNA into the plasmid vector
2. The recombinant plasmid is introduced into a bacterium through transformation.
3. The bacterium replicates millions of times, creating many copies of plasmid.
4. Lyse the bacteria and collect the purified plasmid.

Question 11

Recombinant DNA can be used to produce clinically relevant proteins. Describe how this works and give two examples.

Answer 11

Some plasmids contain the appropriate genetic elements to force transcription and translation of the inserted DNA fragment. Cells can be forced to produce large quantities of proteins. Examples include insulin, human growth hormone, taxol, cellulose, factor VIII.

Question 12

How does gel electrophoresis work?

Answer 12

1. Cut wells in the gel.
2. Submerge the gel in buffer (maintains pH, contains necessary ions)
3. Apply a current
4. DNA (negatively charged) runs toward the anode (positive end)

Question 13

Gel electrophoresis can separate DNA by size. Smaller molecules run ___ down the gel.

Answer 13

Further

Question 14

What type of gel is used for short DNA molecules (

Answer 14

Polyacrylamide gel

Question 15

What type of gel is used for large DNA molecules (>50,000 base pairs)?

Answer 15

Pulsed-field agarose-gel electrophoresis

Question 16

Complementary nucleic acid strands can bind to each other through nucleic acid ___.

Answer 16

Hybridization

Question 17

How is DNA denatured?

Answer 17

Heat or high pH (alkali solution)

Question 18

What is Southern blotting?

Answer 18

Combines electrophoresis and hybridization to detect a specific DNA molecule within a complex mixture.

Question 19

How does Northern blotting differ from Southern blotting?

Answer 19

Northern blotting is for detecting RNA sequences

Question 20

What are probes?

Answer 20

Single-stranded DNA molecules used to detect complementary sequences; carry radioactive or chemical markers to facilitate detection

Question 21

Describe the Southern Blot analysis process.

Answer 21

1. Cleave DNA with a restriction enzyme
2. Run unlabeled DNA on agarose gel to separate by size.
3. Separated nucleic acids are blotted onto nitrocellulose paper by suction of buffer through gel and paper (transferred to a membrane)
4. Remove nitrocellulose paper with bound nucleic acids
5. Radiolabeled probes are hybridized to separated DNA
6. Labeled probes hybridized to complementary DNA bands are visualized.

Question 22

What is RFLP?

Answer 22

Restriction Fragment Length Polymorphism

Question 23

Sickle-cell disease can be detected through ___.

Answer 23

RFLP

Question 24

In sickle-cell disease, Glu mutates to Val, which destroys ____.

Answer 24

A restriction enzyme cut site

Question 25

How can RFLP be used to determine the presence of sickle-cell mutation?

Answer 25

Because the restriction enzyme cut site is removed, the mutated gene will be longer than the unmutated gene.

Question 26

What does in situ hybridization do?

Answer 26

Locates specific DNAs within cells and genes on chromosomes.

Question 27

Describe the process of FISH (Fluorescent in situ hybridization).

Answer 27

1. Denature DNA to create single-stranded DNA. 2. Bind fluorescent to complementary sequences 3. Allows for visualization of multiple probes simultaneously

Question 28

FISH is utilized in the clinical lab for the diagnosis of ___.

Answer 28

Her2 positive breast cancer

Question 29

What is PCR?

Answer 29

Polymerase Chain Reaction; process of amplifying DNA through repeated rounds of synthesis in vitro

Question 30

What are the reactants in PCR?

Answer 30

1. DNA 2. Nuceteotides 3. DNA polymerase 4. PCR primers 5. Buffers/MgCa

Question 31

Describe the steps of PCR.

Answer 31

1. Denature DNA sample to separate DNA strands (denaturation) 2. Primers bind to DNA strands (annealing) 3. Polymerase synthesize new DNA strands (extension)

Question 32

PCR is important for detecting genetic differences between individuals and for detecting disease-causing genes. ___ can be diagnosed with PCR.

Answer 32

Hemophilia

Question 33

Describe diagnosis of hemophilia by PCR.

Answer 33

Hemophilia is a sex-linked disorder (gene for Factor VIII on X-chromosome) with a mutation in intron 18, which removes a restriction enzyme cutting site. Amplification of normal gene is cleaved into 2 fragments; amplification of the mutant gene is only 1 fragment.

Question 34

Describe diagnosis of DMD by PCR.

Answer 34

There are 9 exon regions commonly deleted in DMD. PCR primers designed to these regions give different product sizes. Running a gel with the products shows whether or not bands are missing (missing bands indicate deletion of exons).

Question 35

How can PCR be used in forensic science and paternity tests to identify the source of a DNA sample and distinguish between individuals?

Answer 35

DNA sequences involve short tandem repeats (STRs or microsatellites) composed of 2-6 base pair sequences. These are located in non-coding regions. Because of the variability of repeats, individuals usually inherit a different number of repeats at each STR locus from their mother and from their father. Two unrelated individuals rarely contain the same pair of sequences at a given locus. PCR using primers to recognize unique sequences on either side of one locus produce a pair of bands of amplified DNA from each individual (one for the maternal STR variant and one for the paternal STR variant)

Question 36

What is Reverse Transcriptase PCR used for?

Answer 36

Detecting RNA templates

Question 37

Reverse transcriptase produces ___ from RNA.

Answer 37

Complementary DNA (cDNA)

Question 38

Why can't RNA be used as a PCR template?

Answer 38

It is single stranded and relatively unstable

Question 39

Describe the process of synthesizing cDNA.

Answer 39

1. Total mRNA extracted from tissue. 2. Short oligonucleotide complementary to the poly-A tail at the 3' end of the mRNA (poly-T primer) hybridized to the RNA to act as a primer for reverse transcriptase. 3. Reverse trasncriptase copies the RNA into a cDNA chain, forming a DNA-RNA hybrid helix. 4. Degrade RNA with RNAse H (specialized nuclease) that attacks only RNA. 5. DNA polymerase copies the remaining single-stranded cDNA into double-stranded cDNA. Note that because DNA polymerase can synthesize through the bound RNA molecules, the RNA fragment that is base paired to the 3' end of the first strand usually acts as a primer for the second strand.

Question 40

Reverse-transcriptase PCR can be used to diagnose ___.

Answer 40

HIV

Question 41

What is used to measure the expression of many genes at the same time?

Answer 41

Microarrays

Question 42

Describe the process of using a microarray.

Answer 42

1. Isolate mRNA from a tissue sample. 2. Reverse transcribe mRNA to cDNA using fluorescently labeled nucleotides. 3. Apply cDNA to microarray, which hybridizes with any complementary DNA on the array. A microarray bears copies of single-stranded DNA fragments representing an organisms genes. 4. Wash to remove unbound cDNA. Scan for fluorescence. Each spot represents a gene expressed in the tissue sample.

Question 43

DNA microarrays can be used in the clinic to measures mRNA expression levels for 21 genes in patients with ___.

Answer 43

Estrogen receptor-positive, node-negative breast cancer

Question 44

In traditional sequencing, what happens?

Answer 44

A complementary strand of DNA is synthesized from a single strand DNA template.

Question 45

Describe Sanger sequencing.

Answer 45

1. Denature the DNA into separate strands. 2. Primers bind to DNA strands. 3. Polymerase synthesizes new DNA using labeled dideoxyribonucleotides (dNTPs). These are readily incorporated into a growing chain, but act as chain terminators after incorporation. 4. A terminator will be inserted at every base throughout the sequence. 5. The sequencing reaction is run on a PAGE gel to separate the fragments based on size. Note that the results give the sequence of the complementary strand, not the template strand.

Question 46

DNA sequencing can be used to diagnose genetic diseases caused by changes in DNA sequences, such as ___.

Answer 46

Cystic fibrosis

Question 47

CF is commonly caused by a three-nucleotide deletion in the gene for ___.

Answer 47

CFTR (Cystic fibrosis transmembrane regulator, a chloride ion transporter)

Question 48

Compare traditional sequencing and NGS (next generation sequencing)

Answer 48

1. NGS is faster because the chemical reaction is combined with signal detection and it generates data from thousands of reaction simultaneously. 2. NGS is more accurate because it relies on reading short overlapping fragments, leading to multiple sequencing. NGS has a greater depth of coverage. 3. NGS requires less DNA. 4. NGS is more cost effective in the long run.