Hybridization-uses a labeled(useally radioactive) DNA sequence of interest(probe)

to find a complementary match Fig9.11

-can be done in a gel

-Southern, Northern blotting

-can use samples from growing colonies on a plate
-can be done inside cells on a small scale

for determing if a particular DNA sequence is present can be used with restriction map to indicate where a DNA
sequence is located
Southern Blot

DNA fragments separated by electrophoresis

transfer DNA from gel to a solid membrane support (nitrocellulose on nylon)
Denature DNA(separate strands) temp w/ NaOH
cross link DNA to membrane-bake or use UV
add

fig 9.11b

washing will eliminate nonspecific binding

allow membrane to dry and expose to x-ray film

Northern Blotting
RNA (usually mRNA fragments are separeated by electrophoresis detected with DNA probe
can indicate transcription

useful for performing comparison studies

ex. examine if a gene is transcribed in liver vs brain

Western blot

proteins separated by electrophoreisis

use labeled antibody to indicate the presence of desired protein.
PCR

polymerase chain reaction

inveted by Kary Mullis

very seneitive technique for amplifying particular DNA sequences--> within 2 hours, a single short sequence of
DNA can be copied over 1 million times

Often used for preparation of large amounts of DNA for oher analysis
can be used directly for many other applications

ex. a minute tissue sample at a crime scene can be amplifede before more analysis

 can also used for medical diagnosis

RT-PCR-> "real time" "reverse transcriptase" sometimes both

much more sensitive than other techniques

reverse transcriptase PCR can detect minute quantities of RNA

Primers in PCR are critical Fig.9.12

primers mark ends of the region that will be amplified

essential that primers will only match one position in an entire genome of DNA.
primers usually 20-30 nt long (often close to 30)

longer primers reduce the chances of a close match in another region of DNA

the two primers are usually very similar in G-C content higher G-C content=higher annealing temp
the primers should also begin and end with G or C

PCR involves a repetition of 3 different reactions, each reaction will occur at a different tempeture
1) DNA melting(denaturing)
-94 C

first round-5 minutes

subsequent rounds-20 to 30 seconds

2) Primer annealing

temp varies according to the selected primers, but usually -50-60 C

want a tempeture high enough to prevenet primers from binding other similar sequences
want a temperture low enough to ensure good binding to target DNA
-30 seconds to get good pairing between primers and target DNA
3) DNA synthesis (primer extension)

elevate temp to ideal temp for the DNA polymerase used

Taq polymerase-isolated from a bacterium Themus aquaticus

Fig 9.12
Fig 9.13a

DNA sequencing

Sanger sequencing developed in the 70's but still used today process involves syntheisis of labeled DNA
fragments of Diffrent sizes that can be separated by electrophoresis
most nucleic acid electrophoresis uses agarose gel

agarose good for separating fragments ranging from -15kb to 1kb

most gels are 0.8 to 1% agarose

increasing agarose concentration can separate smaller fragments better, but still limited

 electrophoresis for DNA sequencing uses polyacrylamide gel

ideally suited to separating very small fragments generated in a DNA sequencing reaction
Orginal Sanger sequencing

uses dideoxyNTP chain termination

ddNTP doesn't have a -OH on the 3' and 2' while dNTP lacks on 2' on -OH

the ddNTP chain termination will synthesisze DNA fragments, that are very slightly different in size.
process starts with 4 different reaction tubes

each reaction tube contains a small amount of 1 of the ddNTPs.

each of the reaction tubes contain otherwise similar contents
template DNA

radioactive primer
DNA polymerase

required cofactors
all 4 dNTPs (present in much higher concentration than ddNTP)
Fig. 9.13b

Fig 9.13 c

problems with original Sanger sequencing

- reading sequence e end of the process

-sequence manually read from autoradiograms, diffucult error-prone and time consuming
-prep was very involved also very time-consuming