Amanda Barrera Biology Honors Period 1 DNA Sequencing

What is DNA Sequencing? DNA Sequencing is the process of determining the order of base pairs in a section of DNA.

The Process First, the DNA is unwound and made into two separate strands. These strands are put into four buffer solutions. The buffer solutions have free-floating Nucleotides and the enzyme DNA Polymerase to rebuild the double helix. These buffers also include a Dideoxy Nucleotide, which prevents the helix from being made any further.

The Process For example, in the buffer with the Guanine Dideoxy Nucleotide, the strands are built to every possible length that ends with that nucleotide. The same is for the buffers that contain the other three Dideoxy Nucleotides.

The Process: Electrophoresis The buffer solutions are loaded into wells in an agarose gel.

The Process: Electrophoresis The nucleotides are negatively charged. When electricity is sent through the gel, the nucleotides travel toward the positive side of the gel.

The Process: Electrophoresis The smaller, lighter nucleotides travel faster and farther than larger nucleotides through the cell. After several hours of allowing the electricity to flow through the gel, the gel is then taken out.

The Process: Electrophoresis The gel is then placed under an ultraviolent light, where the radioactive nucleotides can be seen. The shorter nucleotides will appear farther down the gel than longer Nucleotides. The DNA Sequence is read from bottom to top.

DNA Hybridization DNA Hybridization: Process where two complimentary strands of DNA (or a DNA and a RNA strand) come together to form a double helix. The DNA/RNA strand that is used to locate complimentary sequences is called a probe.

DNA Hybridization If the probe is labeled, (by making it radioactive or fluorescent) the complimentary strand it hybridizes to will also become labeled and can be detected. First, the DNA must be separated into two single strands, also known as denaturing, which can be accomplished by heating up the DNA Then, the DNA strand pairs up with the complimentary strands in it’s mixture, including probes.

Southern Blot Southern Blot: Process used for identifying and measuring the amount of a specific DNA in a mixture. First, Electrophoresis occurs. Then, the DNA is transferred onto a nylon filter paper. Once transferred, the radioactive probe solution is added. The probes bind with any complimentary strands.

Southern Blot To identify where the probes are binded, the nylon filter is covered with an X-Ray film. After developing, The probe’s location(s) are shown.

The Sanger Method First the DNA must be separated into two strands. The strand that is going to be sequenced is then copied by using chemically altered bases. The bases stop the copying process at every possible length that ends with that nucleotide. This process is continued for all four bases. The fragments are then put together to reveal the base sequence of the DNA strand.

Applications: DNA Forensics DNA Fingerprinting(forensics): Process that uses PCR and Gel Electrophoresis to Examine DNA. PCR (Polymerase Chain Recation): Process that uses a heat-stable DNA Polymerase to copy desired genes repeatedly. Used in Crime Scene Investigations.

Applications: DNA Forensics (PCR) CYCLE 1: First, heat up the DNA to separate the helix. Cool down to allow premade primers (that are made to specifically bind to the 3’ side of the target sequence of DNA). Free nucleotides attach to DNA while Polymerase Copies the Target Section of DNA starting from the primer. Creates 2 longer length copies.

Applications: DNA Forensics (PCR) CYCLE 2: Repeat procedure done in Cycle 1 with new strands. Creates 4 longer length copies. CYCLE 3: Repeat separating process from previous cycles with new strands(some are only the length of the target gene) Repeat remaining steps from previous cycles. Creates 2 target genes and 6 longer length copies.

Applications: DNA Forensics (PCR) CYCLE 4: Repeat processes from previous cycles with new strands. Creates 8 longer length strands and 8 target strands. CYCLE 5: Creates 10 longer length strands and 22 target strands after repeating processes from previous cycles. Cycles continue until roughly a billion target strands are created.

Applications: DNA Forensics (Electrophoresis) Electrophoresis occurs. Data is evaluated as usual. DNA Forensics can be used to evaluate genetic risks along with who a suspect is.

“That’s all Folks!” DNA Sequencing By: Amanda Barrera Biology Honors Period 1 Thanks For Watching!