Molecular Biology Techniques

Question 1

DNA techniques, clinical applications and emerging applications

Answer 1

• Restriction Enzyme Digestion, Southern Blotting, conventional sequencing & PCR
• Identification of mutations and infectious agents
• Prenatal screening

Question 2

mRNA techniques, clinical applications and emerging applications

Answer 2

• Northern Blotting, RT-PCR, microarray analysis
• Breast cancer stratification
• occular melanoma & breast cancer subtypes

Question 3

Protein techniques, clinical applications and emerging applications

Answer 3

• ELISA & Western blotting
• Diagnosis of infectious diseases
• Therapeutic antibodies

Question 4

Emerging molecular techniques:

Answer 4

“Next generation” sequencing & RNA Interference

Question 5

Restriction enzyme digestion:

Answer 5

*Used to ID DNA
*Used to recombine DNAs
Restriction enzyme binds and makes a DOUBLE-stranded cut creating a characteristic set of fragments, which can be used to identify pieces of DNA. (“restriction enzyme digestion”.)
100’s of enzymes recognizing different sites are known making restriction enzymes extremely useful for molecular biology.
EcoR1 recognizes and binds to the double stranded (ds) sequence 5’ GAATTC 3’ generating 4 BP-long single stranded ends - known as sticky ends because they can rehybridize to each other or to any other piece of DNA cut with same enzyme.

Question 6

How do DNA sequences create unique protein binding sites?

Answer 6

Sequence –> unique H-bonding sites in Major grooves for things like transcription factors & restriction enzymes.

Question 7

How is restriction enzyme digestion able to ID DNA being cut?

Answer 7

Digestion of a DNA sequence with a restriction enzyme gives a specific set of fragments. Gel electrophoresis can then be used to separate segments by size. Since DNA has a negative charge, when an electrical field is applied all fragments migrate, but the small fragments move faster and, thus, farther than the large. The segments can then be stained and visualized.

Question 8

True or false, gel electrophoresis is only used in DNA separation.

Answer 8

False, it’s also used for separating RNA & proteins by size. SDS can be used to first neutralize protein charge.

Question 9

Southern Blotting

Answer 9

• Used to ID a specific region of DNA.
• Target DNA is ID’d based on restriction fragment SIZE plus hybridization to a PROBE.
• A target DNA in a complex mixture, i.e. one gene in the entire genome, can be identified.

Question 10

Southern Blotting steps:

Answer 10

1. Genomic DNA is digested with a restriction enzyme.
2. Fragments are separated by size on an agarose gel.
3. DNA is denatured and transferred to a membrane.
4. Membrane is incubated with a probe.
   * Probe: Defined piece of DNA homologous to target that is labelled with radioactivity or fluorescence for detection.
5. Probe DNA is denatured and added to membrane.
6. If DNA sequence encoded in the probe is present in the DNA mixture, then complementary strands of probe and target DNA will bind to each other.
7. Sites of probe binding are visualized by radioactive or fluorescent tag on probe binding, which identifies sites where target DNA is present.

Question 11

Sanger or conventional DNA Sequencing

Answer 11

1. The DNA to be sequenced is denatured to provide a template for synthesis of a new strand. 2. Purified dNTPs are provided.
2. The pool of normal dNTPs is spiked with a very low concentration of fluorescently-labeled ddNTP (such as ddTTP). Every where that an A is (for T ddTTP to bind) lights up.
   This occurs because ddTTP lacks a 3’ OH necessary for incoming dNTP’s to bind causing termination.
3. Four sequencing reactions are required - one for each nt.
4. All the fragments are separated by gel electro.
5. Size of fragments corresponds to position of ddNTP in the sequence
6. Position of fragments in gel corresponds to position of ddNTP in DNA sequence

Question 12

Polymerase Chain Reaction (PCR)

Answer 12

• PCR AMPLIFIES a defined region of DNA *Extremely powerful b/c combines specificity AND amplification
• Amplification by repeated cycles of in vitro DNA replication
• Specificity because region to be amplified is determined by hybridization of short primers

Question 13

Steps in each PCR cycle:

Answer 13

1. DNA is denatured, two strands separated
2. Primers anneal flanking target region by complementary base pairing.
3. Primers prime synthesis of complementary strands of target DNA in 5’ to 3’ direction.
4. In the next round each of the new double stranded DNAs serve as templates for further amplification.
5. End result is exponential amplification of starting DNA

Question 14

PCR is an example of in vitro _____.

Answer 14

DNA synthesis

Question 15

In PCR, _____determine the specific region of DNA that will be amplified.

Answer 15

Primers. Two are used so that both strands are replicated.

Question 16

Clinical applications of DNA techniques

Answer 16

*Diagnosis of infectious diseases-
HPV, human papillomavirus:
–Some strains are causative agents for cervical cancer.
–Kit based on hybridization of RNA probes to viral DNA
–Probes are designed to be strain specific when used as screening tool

*Identification of mutations that cause hereditary diseases and risk factors-
BRCA1 and BRCA2:
–BRCA1 and BRCA2 mutations are by far the most prevalent breast cancer genetic risk factors
–Commercially available test by DNA sequencing

Question 17

EMERGING clinical application of DNA techniques: molecular prenatal diagnosis

Answer 17

Prenatal testing is routinely offered for trisomy 21, but it’s fairly invasive. Alternate approach: analyze fetal DNA in maternal blood to determine if levels of chromosome 21 are elevated. Maternal serum has both fetal and maternal cell free DNA. *If fetus has trisomy 21, maternal serum will have higher ratio of chromosome 21:chromosome 12.

• Problem- concentration of fetal DNA is very low
• **Solution - Digital PCR, extremely sensitive, is being developed to measure small excess DNA

Question 18

_______ is based on DNA-RNA binding, or hybridization, through complementary base pairing.

It is used to identify one target mRNA in a complex mixture, ie all RNAs expressed in a cell.

Answer 18

Northern blotting

Question 19

________ adapts PCR to quantitatively measure mRNA levels. What are the steps in this process?

Answer 19

RT-PCR (reverse transcriptase-PCR)
1. mRNA is converted to cDNA by reverse transcriptase.
2. Specific primers are used with PCR.
3. Fluorescent dye is incorporated as new
DNA is synthesized & amplified.
**The result is that the intensity of the fluorescence is proportional to the amount of mRNA initially present.

Question 20

Why is mRNA converted to cDNA in RT-PCR?

Answer 20

RNA not a template for most DNA polymerases

Question 21

Microarray analysis

Answer 21

Microarray analysis is used to determine global patterns of mRNA expression. The levels of 1000s of individual mRNAs are measured simultaneously. This is an extremely powerful technique which can identify individual genes whose expression changes in disease states. And possibly even more important it can identify patterns of mRNAs expressed in different diseases.

Question 22

Microarray technique summary:

Answer 22

Based on hybridization of a cDNA population derived from sample mRNAs to a grid containing known DNAs at specific locations.

Question 23

Microarray process:

Answer 23

Make array on chip:
1000s of short DNAs (oligos), 25 bases long.
Derived from genes to be analyzed
Arranged in grid- sequence, corresponding
gene and location for each oligo is known.
Up to 40,000 genes per chip!!!

Question 24

Microarray methodology:

Answer 24

• mRNA population in patient’s cells are compared to mRNAs in reference sample
• cDNAs derived from patient and reference mRNA populations are labelled with different fluorescent tags
• Tagged cDNAs derived from original mRNAs are hybridized to an array of oligos representing mRNAs to be interrogated
• Pattern of fluorescence on chip tells which mRNAs are present in each population and relative levels

Question 25

Microarray data analysis

Answer 25

• Tells which mRNAs are present in each original sample
• Tells relative levels of each mRNA in original samples- patient vs. reference
• Defines signature for each patient sample
• Does NOT tell which mRNA changes “cause” the disease

Question 26

Clinical applications of RNA techniques

Answer 26

RT-PCR or RT-PCR+sequencing-

• -Identification of genetic alterations that affect protein coding regions
• OTC used to ID fusion genes in leukemias and lymphomas
• -ID of infectious agents
• Screening blood pool or HIV

Microarray analysis-
*Oncotype DX- widely used test to determine risk of recurrence in breast cancer. Some results show tumors that would recur.

Question 27

Emerging RNA clinical application of RNA techniques: cancer classification

Answer 27

Identifying different sub-groups among cancer patients and tailoring therapies accordingly.

Question 28

The foundation of most protein characterization techniques is the specific interaction
between an _____ and a protein.

Answer 28

antibody

Question 29

Natural function of antibodies:

Answer 29

*Antibodies are proteins made by B cells as part of the immune response.
*Antibodies bind foreign proteins and tag them for processing by the immune system.
*An individual has the potential to make an almost infinite number of different antibodies
with different binding sites.

Question 30

Antibody structure

Answer 30

Variable regions- differ among antibodies,
bind specific proteins (ends of “Y”)
Constant regions- constant among same
class of antibodies (The stem of the “Y”)

Question 31

_______ recognizes specific target protein in protein testing.

Answer 31

Primary antibody

Question 32

______ has tag that emits signal when activated and recognizes constant region of primary antibody. True/false, these can be used for all primary antibodies with same constant region.

Answer 32

Secondary antibody

Question 33

ELISA- enzyme linked immunosorbent assay

Answer 33

Used to detect specific protein in complex mixture
High throughput and quantitative
Used extensively clinically

Also based on specific interaction of protein with antibody, but NO separation by size (so not as specific as W Blot)

Question 34

How does ELISA work? Use HIV as an example.

Answer 34

Recombinant HIV proteins are immobilized on agarose beads or on the wells of a 96 well plate. These proteins serve as bait for HIV antibodies in the patient’s serum. If the patient’s serum contains HIV antibodies, they will bind to the immobilized HIV protein antigen and also become immobilized. Unbound antibodies will be washed away. Bound antibodies are detected using a secondary antibody conjugated to an enzyme which causes a substrate to change color. The color change is detected and quantitated by an automatic plate reader. Darker color means higher [antibody].

Question 35

Western blotting

Answer 35

• Identifies a specific protein in a complex mix, ie total cell lysate
• Target protein is identified by size AND specific interaction between antibody and protein…similar to Southern and Northern Blotting, but looking at proteins
• -Size fractionation of protein mixture
• -Specific binding of antibody to target protein
• -Detection using secondary antibody

Comparable to Southern and Northern blotting

Question 36

How western blotting works

Answer 36

1. Preparation of protein - protein is denatured and incubated with SDS to give a uniform mass to charge ratio so that migration depends on mass not native charge or shape.
2. Proteins are size fractionated on a polyacrylamide gel (SDS-PAGE).
3. Proteins are transferred to a membrane.
4. Membrane is incubated with primary antibody specific for target protein.
5. The position of he primary antibody and target protein are visualized using a secondary antibody. The secondary antibody binds to the conserved constant region so that the same secondary can be used for many primaries. The secondary antibody is conjugated to an enzyme that cleaves a substrate to release light. Signal is detected only at the site of the target protein.

Question 37

Significance of Western Blotting:

Answer 37

Western blotting is essential for research to identify changes in protein expression and modification during pathogenesis but it is labor intensive and difficult to quantitate so is not commonly used as a first round test.

Question 38

Emerging application: therapeutic antibodies

Answer 38

Because of specificity antibodies have great potential as therapeutic anti-cancer agents. By blocking cell surface receptor, antibodies could evoke desirable immune responses, deliver toxin or radioactive material to kill cancer cells, for example.

Question 39

Herceptin and Her2

Answer 39

HER2 is a member of the epidermal growth factor receptor family. Overexpression of HER2 –> 25% of breast cancers. Herceptin blocks HER2 growth factor receptor

Question 40

Next Generation Sequencing, aka massively parallel, deep sequencing:

Answer 40

massively parallel- make millions of short fragments and sequenced in one run
quantitative- digital readout gives number of fragments with a given sequence
IOW - it’s waaaaay faster and gives more information than conventional/Sanger sequencing.

Question 41

Next Generation Sequencing Goals & Limitations:

Answer 41

• Identify mutations or genetic variants that cause inherited diseases or susceptibility to diseases
• Identify somatic mutations in cancers
• cost still high
• interpretation- which sequence differences are important
• **Most limiting - effective treatments are often not available ***

Question 42

RNA interference, RNAi

Answer 42

Small non-coding RNAs regulate gene expression in the cell by targeting specific mRNAs.E.g. microRNA (miRNA) & Short interfering RNA (siRNA)

Clinical potential is to specifically deplete
mRNAs involved in disease by synthesizing miRNA and getting it to target tissues.